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Sample records for identify biochemical pathways

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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 Nε-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

  6. 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

  7. Identifying Branched Metabolic Pathways by Merging Linear Metabolic Pathways

    NASA Astrophysics Data System (ADS)

    Heath, Allison P.; Bennett, George N.; Kavraki, Lydia E.

    This paper presents a graph-based algorithm for identifying complex metabolic pathways in multi-genome scale metabolic data. These complex pathways are called branched pathways because they can arrive at a target compound through combinations of pathways that split compounds into smaller ones, work in parallel with many compounds, and join compounds into larger ones. While most previous work has focused on identifying linear metabolic pathways, branched metabolic pathways predominate in metabolic networks. Automatic identification of branched pathways has a number of important applications in areas that require deeper understanding of metabolism, such as metabolic engineering and drug target identification. Our algorithm utilizes explicit atom tracking to identify linear metabolic pathways and then merges them together into branched metabolic pathways. We provide results on two well-characterized metabolic pathways that demonstrate that this new merging approach can efficiently find biologically relevant branched metabolic pathways with complex structures.

  8. Construction and engineering of large biochemical pathways via DNA assembler

    PubMed Central

    Shao, Zengyi; Zhao, Huimin

    2015-01-01

    Summary DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated. PMID:23996442

  9. Modeling biochemical pathways in the gene ontology

    PubMed Central

    Hill, David P.; D’Eustachio, Peter; Berardini, Tanya Z.; Mungall, Christopher J.; Renedo, Nikolai; Blake, Judith A.

    2016-01-01

    The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. PMID:27589964

  10. Modeling biochemical pathways in the gene ontology.

    PubMed

    Hill, David P; D'Eustachio, Peter; Berardini, Tanya Z; Mungall, Christopher J; Renedo, Nikolai; Blake, Judith A

    2016-01-01

    The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. PMID:27589964

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

    PubMed

    Cherry, J Michael

    2015-12-01

    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

  12. Using Bioinformatic Approaches to Identify Pathways Targeted by Human Leukemogens

    PubMed Central

    Thomas, Reuben; Phuong, Jimmy; McHale, Cliona M.; Zhang, Luoping

    2012-01-01

    We have applied bioinformatic approaches to identify pathways common to chemical leukemogens and to determine whether leukemogens could be distinguished from non-leukemogenic carcinogens. From all known and probable carcinogens classified by IARC and NTP, we identified 35 carcinogens that were associated with leukemia risk in human studies and 16 non-leukemogenic carcinogens. Using data on gene/protein targets available in the Comparative Toxicogenomics Database (CTD) for 29 of the leukemogens and 11 of the non-leukemogenic carcinogens, we analyzed for enrichment of all 250 human biochemical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The top pathways targeted by the leukemogens included metabolism of xenobiotics by cytochrome P450, glutathione metabolism, neurotrophin signaling pathway, apoptosis, MAPK signaling, Toll-like receptor signaling and various cancer pathways. The 29 leukemogens formed 18 distinct clusters comprising 1 to 3 chemicals that did not correlate with known mechanism of action or with structural similarity as determined by 2D Tanimoto coefficients in the PubChem database. Unsupervised clustering and one-class support vector machines, based on the pathway data, were unable to distinguish the 29 leukemogens from 11 non-leukemogenic known and probable IARC carcinogens. However, using two-class random forests to estimate leukemogen and non-leukemogen patterns, we estimated a 76% chance of distinguishing a random leukemogen/non-leukemogen pair from each other. PMID:22851955

  13. Simulation of Biochemical Pathway Adaptability Using Evolutionary Algorithms

    SciTech Connect

    Bosl, W J

    2005-01-26

    The systems approach to genomics seeks quantitative and predictive descriptions of cells and organisms. However, both the theoretical and experimental methods necessary for such studies still need to be developed. We are far from understanding even the simplest collective behavior of biomolecules, cells or organisms. A key aspect to all biological problems, including environmental microbiology, evolution of infectious diseases, and the adaptation of cancer cells is the evolvability of genomes. This is particularly important for Genomes to Life missions, which tend to focus on the prospect of engineering microorganisms to achieve desired goals in environmental remediation and climate change mitigation, and energy production. All of these will require quantitative tools for understanding the evolvability of organisms. Laboratory biodefense goals will need quantitative tools for predicting complicated host-pathogen interactions and finding counter-measures. In this project, we seek to develop methods to simulate how external and internal signals cause the genetic apparatus to adapt and organize to produce complex biochemical systems to achieve survival. This project is specifically directed toward building a computational methodology for simulating the adaptability of genomes. This project investigated the feasibility of using a novel quantitative approach to studying the adaptability of genomes and biochemical pathways. This effort was intended to be the preliminary part of a larger, long-term effort between key leaders in computational and systems biology at Harvard University and LLNL, with Dr. Bosl as the lead PI. Scientific goals for the long-term project include the development and testing of new hypotheses to explain the observed adaptability of yeast biochemical pathways when the myosin-II gene is deleted and the development of a novel data-driven evolutionary computation as a way to connect exploratory computational simulation with hypothesis

  14. Proteomic Assessment of Biochemical Pathways That Are Critical to Nickel-Induced Toxicity Responses in Human Epithelial Cells.

    PubMed

    Ge, Yue; Bruno, Maribel; Haykal-Coates, Najwa; Wallace, Kathleen; Andrews, Debora; Swank, Adam; Winnik, Witold; Ross, Jeffrey A

    2016-01-01

    Understanding the mechanisms underlying toxicity initiated by nickel, a ubiquitous environmental contaminant and known human carcinogen is necessary for proper assessment of its risks to human and environment. Among a variety of toxic mechanisms, disruption of protein responses and protein response-based biochemical pathways represents a key mechanism through which nickel induces cytotoxicity and carcinogenesis. To identify protein responses and biochemical pathways that are critical to nickel-induced toxicity responses, we measured cytotoxicity and changes in expression and phosphorylation status of 14 critical biochemical pathway regulators in human BEAS-2B cells exposed to four concentrations of nickel using an integrated proteomic approach. A subset of the pathway regulators, including interleukin-6, and JNK, were found to be linearly correlated with cell viability, and may function as molecular determinants of cytotoxic responses of BEAS-2B cells to nickel exposures. In addition, 128 differentially expressed proteins were identified by two dimensional electrophoresis (2-DE) and mass spectrometry. Principal component analysis, hierarchical cluster analyses, and ingenuity signaling pathway analysis (IPA) identified putative nickel toxicity pathways. Some of the proteins and pathways identified have not previously been linked to nickel toxicity. Based on the consistent results obtained from both ELISA and 2-DE proteomic analysis, we propose a core signaling pathway regulating cytotoxic responses of human BEAS-2B cells to nickel exposures, which integrates a small set of proteins involved in glycolysis and gluconeogenesis pathways, apoptosis, protein degradation, and stress responses including inflammation and oxidative stress. PMID:27626938

  15. Deterministic modelling and stochastic simulation of biochemical pathways using MATLAB.

    PubMed

    Ullah, M; Schmidt, H; Cho, K H; Wolkenhauer, O

    2006-03-01

    The analysis of complex biochemical networks is conducted in two popular conceptual frameworks for modelling. The deterministic approach requires the solution of ordinary differential equations (ODEs, reaction rate equations) with concentrations as continuous state variables. The stochastic approach involves the simulation of differential-difference equations (chemical master equations, CMEs) with probabilities as variables. This is to generate counts of molecules for chemical species as realisations of random variables drawn from the probability distribution described by the CMEs. Although there are numerous tools available, many of them free, the modelling and simulation environment MATLAB is widely used in the physical and engineering sciences. We describe a collection of MATLAB functions to construct and solve ODEs for deterministic simulation and to implement realisations of CMEs for stochastic simulation using advanced MATLAB coding (Release 14). The program was successfully applied to pathway models from the literature for both cases. The results were compared to implementations using alternative tools for dynamic modelling and simulation of biochemical networks. The aim is to provide a concise set of MATLAB functions that encourage the experimentation with systems biology models. All the script files are available from www.sbi.uni-rostock.de/ publications_matlab-paper.html. PMID:16986253

  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. 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

  18. LeishCyc: a biochemical pathways database for Leishmania major

    PubMed Central

    Doyle, Maria A; MacRae, James I; De Souza, David P; Saunders, Eleanor C; McConville, Malcolm J; Likić, Vladimir A

    2009-01-01

    Background Leishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen. Description The LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2), based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute). Conclusion The LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania biochemical networks and is

  19. 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…

  20. 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

  1. Characterization of changes in gene expression and biochemical pathways at low levels of benzene exposure.

    PubMed

    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

  2. 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

  3. 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.

  4. Genetic variants in the Hippo pathway predict biochemical recurrence after radical prostatectomy for localized prostate cancer.

    PubMed

    Huang, Chao-Yuan; Huang, Shu-Pin; Lin, Victor C; Yu, Chia-Cheng; Chang, Ta-Yuan; Juang, Shin-Hun; Bao, Bo-Ying

    2015-01-01

    While localized prostate cancer is potentially curative, many patients still show biochemical recurrence (BCR) after curative treatments such as radical prostatectomy (RP). The Hippo pathway has recently been shown to be an evolutionarily conserved regulator of tissue growth, and its perturbation can trigger tumorigenesis. We hypothesize that genetic variants of the Hippo pathway may influence clinical outcomes in localized prostate cancer patients. We genotyped 53 tagging single-nucleotide polymorphisms (SNPs) from seven core Hippo pathway genes in 246 localized prostate cancer patients treated with RP. Kaplan-Meier analysis and Cox proportional hazard models were utilized to identify significant SNPs that correlated with BCR. For replication, five associated SNPs were genotyped in an independent cohort of 212 patients. After adjusting for known clinicopathologic factors, the association between STK3 rs7827435 and BCR (P = 0.018) was replicated in the second stage (P = 0.026; Pcombined = 0.001). Additional integrated in silico analysis provided evidence that rs7827435 affects STK3 expression, which in turn is significantly correlated with tumor aggressiveness and patient prognosis. In conclusion, genetic variants of the Hippo pathway contribute to the variable outcomes of prostate cancer, and the discovery of these biomarkers provides a molecular approach for prognostic risk assessment. PMID:25707771

  5. Fungal Indole Alkaloid Biosynthesis: Genetic and Biochemical Investigation of Tryptoquialanine Pathway in Penicillium aethiopicum

    PubMed Central

    Gao, Xue; Chooi, Yit-Heng; Ames, Brian D.; Wang, Peng; Walsh, Christopher T.; Tang, Yi

    2011-01-01

    Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline 2 that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine 1, which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual oxidative opening of the pyrazinone ring by an FAD-dependent berberine bridge enzyme-like oxidoreductase has been proposed based on genetic knockout studies. Notably, a 2-aminoisobutyric acid (AIB)-utilizing NRPS module has been identified and reconstituted in vitro, along with two putative enzymes of unknown functions that are involved in the synthesis of the unnatural amino acid by genetic analysis. This work provides new genetic and biochemical insights into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2. PMID:21299212

  6. Systematic tracking of dysregulated modules identifies disrupted pathways in narcolepsy

    PubMed Central

    Liu, Zhenhua; Zhao, Jiali; Tan, Yinyin; Tang, Minglu; Li, Guanzhen

    2015-01-01

    Objective: The objective of this work is to identify disrupted pathways in narcolepsy according to systematically tracking the dysregulated modules of reweighted Protein-Protein Interaction (PPI) networks. Here, we performed systematic identification and comparison of modules across normal and narcolepsy conditions by integrating PPI and gene-expression data. Methods: Firstly, normal and narcolepsy PPI network were inferred and reweighted based on Pearson correlation coefficient (PCC). Then, modules in PPI network were explored by clique-merging algorithm and we identified altered modules using a maximum weight bipartite matching and in non-increasing order. Finally, pathways enrichment analyses of genes in altered modules were carried out based on Expression Analysis Systematic Explored (EASE) test to illuminate the biological pathways in narcolepsy. Results: Our analyses revealed that 235 altered modules were identified by comparing modules in normal and narcolepsy PPI network. Pathway functional enrichment analysis of disrupted module genes showed 59 disrupted pathways within threshold P < 0.001. The most significant five disrupted pathways were: oxidative phosphorylation, T cell receptor signaling pathway, cell cycle, Alzheimer’s disease and focal adhesion. Conclusions: We successfully identified disrupted pathways and these pathways might be potential biological processes for treatment and etiology mechanism in narcolepsy. PMID:26309600

  7. 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

  8. Systematic tracking of altered modules identifies disrupted pathways in teratozoospermia.

    PubMed

    Huang, Z Q; Wang, G X; Jiang, X L; Tian, E P; Yao, W L; Zeng, T

    2016-01-01

    The objective of this study was to identify disrupted pathways in teratozoospermia by systematically tracking dysregulated modules in reweighted protein-protein interaction (PPI) networks. We inferred and reweighted the PPI networks of normal and teratozoospermia groups based on Spearman correlation coefficients. Modules in the PPI networks were explored via a clique-merging algorithm and altered modules were identified based on maximum weight bipartite matching. Furthermore, pathway-enrichment analyses of genes in altered modules were performed by Database for Annotation, Visualization, and Integrated Discovery (DAVID) to illuminate the biological pathways in teratozoospermia. A total of 20,102 genes were screened from the expression profile. We explored 2406 and 2101 modules in normal and disease PPI networks, respectively. Moreover, we obtained 875 altered modules by comparing modules in normal and teratozoospermia PPI networks. At P < 0.01, the genes involved in 2855 interactions with score changes >1 were mainly enriched in 66 pathways and the genes in altered modules were enriched in 71 pathways. The activity genes (missed and added genes in the disrupted modules) were enriched in 41 common pathways. There were 36 mutual enriched pathways under the five different conditions. Moreover, the cell cycle pathway was disrupted in the first 10 pathways of each condition. This study provides a powerful biomarker discovery platform to better understand the progression of teratozoospermia by systematically tracking dysregulated modules. This method uncovered potential diagnostic and therapeutic targets of teratozoospermia. This information might lead to improved monitoring and treatment of teratozoospermia. PMID:27173237

  9. Using ILP to Identify Pathway Activation Patterns in Systems Biology

    PubMed Central

    Neaves, Samuel R; Millard, Louise A C; Tsoka, Sophia

    2016-01-01

    We show a logical aggregation method that, combined with propositionalization methods, can construct novel structured biological features from gene expression data. We do this to gain understanding of pathway mechanisms, for instance, those associated with a particular disease. We illustrate this method on the task of distinguishing between two types of lung cancer; Squamous Cell Carcinoma (SCC) and Adenocarcinoma (AC). We identify pathway activation patterns in pathways previously implicated in the development of cancers. Our method identified a model with comparable predictive performance to the winning algorithm of a recent challenge, while providing biologically relevant explanations that may be useful to a biologist. PMID:27478883

  10. Physiological and biochemical characteristics of the ethyl tiglate production pathway in the yeast Saprochaete suaveolens.

    PubMed

    Grondin, Eric; Shum Cheong Sing, Alain; Caro, Yanis; de Billerbeck, Gustavo M; François, Jean Marie; Petit, Thomas

    2015-01-01

    A yeast identified as Saprochaete suaveolens was investigated for its capacity to produce a large panel of flavouring molecules. With a production of 32 compounds including 28 esters, S. suaveolens seems to be a good producer of fruity flavours and fragrances and especially of unsaturated esters, such as ethyl tiglate. Physiological and biochemical analyses were performed in this study in an attempt to comprehend the metabolic route to the formation of this compound. We show that the accumulation of ethyl tiglate by S. suaveolens is specifically induced by isoleucine. However, and contrary to S. cerevisiae, which harbours a classical Ehrlich pathway leading to the production of 2-methylbutanol from isoleucine, our results provide phenotypic and enzymological evidence of ethyl tiglate biosynthesis in S. suaveolens through the catabolism of this amino acid by the β-oxidation pathway, which generates tiglyl-CoA as a probable intermediate. A kinetic analysis of this flavour molecule during growth of S. suaveolens on glucose and isoleucine showed a phase of production of ethyl tiglate that culminated concurrently with isoleucine exhaustion, followed by a disappearance of this compound, likely due to reassimilation by the yeast. PMID:25407290

  11. Utilizing signature-score to identify oncogenic pathways of cholangiocarcinoma

    PubMed Central

    Hsiao, Tzu-Hung; Chen, Hung-I Harry; Lu, Jo-Yang; Lin, Pei-Ying; Keller, Charles; Comerford, Sarah; Tomlinson, Gail E.; Chen, Yidong

    2013-01-01

    Extracting maximal information from gene signature sets (GSSs) via microarray-based transcriptional profiling involves assigning function to up and down regulated genes. Here we present a novel sample scoring method called Signature-score (S-score) which can be used to quantify the expression pattern of tumor samples from previously identified gene signature sets. A simulation result demonstrated an improved accuracy and robustness by S-score method comparing with other scoring methods. By applying the S-score method to cholangiocarcinoma (CAC), an aggressive hepatic cancer that arises from bile ducts cells, we identified enriched oncogenic pathways in two large CAC data sets. Thirteen pathways were enriched in CAC compared with normal liver and bile duct. Moreover, using S-score, we were able to dissect correlations between CAC-associated oncogenic pathways and Gene Ontology function. Two major oncogenic clusters and associated functions were identified. Cluster 1, which included beta-catenin and Ras, showed a positive correlation with the cell cycle, while cluster 2, which included TGF-beta, cytokeratin 19 and EpCAM was inversely correlated with immune function. We also used S-score to identify pathways that are differentially expressed in CAC and hepatocellular carcinoma (HCC), the more common subtype of liver cancer. Our results demonstrate the utility and effectiveness of S-score in assigning functional roles to tumor-associated gene signature sets and in identifying potential therapeutic targets for specific liver cancer subtypes. PMID:23905013

  12. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases.

    PubMed

    Lin, Peng-Lin; Yu, Ya-Wen; Chung, Ren-Hua

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn's disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn's disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  13. 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.

  14. Activity-based protein profiling for biochemical pathway discovery in cancer

    PubMed Central

    Nomura, Daniel K.; Dix, Melissa M.; Cravatt, Benjamin F.

    2011-01-01

    Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment. PMID:20703252

  15. Network Modeling Identifies Patient-specific Pathways in Glioblastoma

    PubMed Central

    Tuncbag, Nurcan; Milani, Pamela; Pokorny, Jenny L.; Johnson, Hannah; Sio, Terence T.; Dalin, Simona; Iyekegbe, Dennis O.; White, Forest M.; Sarkaria, Jann N.; Fraenkel, Ernest

    2016-01-01

    Glioblastoma is the most aggressive type of malignant human brain tumor. Molecular profiling experiments have revealed that these tumors are extremely heterogeneous. This heterogeneity is one of the principal challenges for developing targeted therapies. We hypothesize that despite the diverse molecular profiles, it might still be possible to identify common signaling changes that could be targeted in some or all tumors. Using a network modeling approach, we reconstruct the altered signaling pathways from tumor-specific phosphoproteomic data and known protein-protein interactions. We then develop a network-based strategy for identifying tumor specific proteins and pathways that were predicted by the models but not directly observed in the experiments. Among these hidden targets, we show that the ERK activator kinase1 (MEK1) displays increased phosphorylation in all tumors. By contrast, protein numb homolog (NUMB) is present only in the subset of the tumors that are the most invasive. Additionally, increased S100A4 is associated with only one of the tumors. Overall, our results demonstrate that despite the heterogeneity of the proteomic data, network models can identify common or tumor specific pathway-level changes. These results represent an important proof of principle that can improve the target selection process for tumor specific treatments. PMID:27354287

  16. 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

  17. Identifying Critical Pathways to High-Performance PV: Preprint

    SciTech Connect

    Symko-Davies, M.; Noufi, R.; Kurtz, S.

    2002-05-01

    This conference paper describes the High-Performance Photovoltaic (HiPerf PV)Project was initiated by the U.S. Department of Energy to substantially increase the viability of photovoltaics (PV) for cost-competitive applications so that PV can contribute significantly to our energy supply and our environment in the 21st century. To accomplish this, the NCPV directs in-house and subcontracted research in high-performance polycrystalline thin-film and multijunction concentrator devices. Details of the subcontractor and in-house progress will be described toward identifying critical pathways of 25% polycrystalline thin-film tandem cells and developing multijunction concentrator modules to 33%.

  18. A structured approach for the engineering of biochemical network models, illustrated for signalling pathways.

    PubMed

    Breitling, Rainer; Gilbert, David; Heiner, Monika; Orton, Richard

    2008-09-01

    Quantitative models of biochemical networks (signal transduction cascades, metabolic pathways, gene regulatory circuits) are a central component of modern systems biology. Building and managing these complex models is a major challenge that can benefit from the application of formal methods adopted from theoretical computing science. Here we provide a general introduction to the field of formal modelling, which emphasizes the intuitive biochemical basis of the modelling process, but is also accessible for an audience with a background in computing science and/or model engineering. We show how signal transduction cascades can be modelled in a modular fashion, using both a qualitative approach--qualitative Petri nets, and quantitative approaches--continuous Petri nets and ordinary differential equations (ODEs). We review the major elementary building blocks of a cellular signalling model, discuss which critical design decisions have to be made during model building, and present a number of novel computational tools that can help to explore alternative modular models in an easy and intuitive manner. These tools, which are based on Petri net theory, offer convenient ways of composing hierarchical ODE models, and permit a qualitative analysis of their behaviour. We illustrate the central concepts using signal transduction as our main example. The ultimate aim is to introduce a general approach that provides the foundations for a structured formal engineering of large-scale models of biochemical networks. PMID:18573813

  19. 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

  20. Microfluidics meets metabolomics to reveal the impact of Campylobacter jejuni infection on biochemical pathways.

    PubMed

    Mortensen, Ninell P; Mercier, Kelly A; McRitchie, Susan; Cavallo, Tammy B; Pathmasiri, Wimal; Stewart, Delisha; Sumner, Susan J

    2016-06-01

    Microfluidic devices that are currently being used in pharmaceutical research also have a significant potential for utilization in investigating exposure to infectious agents. We have established a microfluidic device cultured with Caco-2 cells, and utilized metabolomics to investigate the biochemical responses to the bacterial pathogen Campylobacter jejuni. In the microfluidic devices, Caco-2 cells polarize at day 5, are uniform, have defined brush borders and tight junctions, and form a mucus layer. Metabolomics analysis of cell culture media collected from both Caco-2 cell culture systems demonstrated a more metabolic homogenous biochemical profile in the media collected from microfluidic devices, compared with media collected from transwells. GeneGo pathway mapping indicated that aminoacyl-tRNA biosynthesis was perturbed by fluid flow, suggesting that fluid dynamics and shear stress impacts the cells translational quality control. Both microfluidic device and transwell culturing systems were used to investigate the impact of Campylobacter jejuni infection on biochemical processes. Caco-2 cells cultured in either system were infected at day 5 with C. jejuni 81-176 for 48 h. Metabolomics analysis clearly differentiated C. jejuni 81-176 infected and non-infected medias collected from the microfluidic devices, and demonstrated that C. jejuni 81-176 infection in microfluidic devices impacts branched-chain amino acid metabolism, glycolysis, and gluconeogenesis. In contrast, no distinction was seen in the biochemical profiles of infected versus non-infected media collected from cells cultured in transwells. Microfluidic culturing conditions demonstrated a more metabolically homogenous cell population, and present the opportunity for studying host-pathogen interactions for extended periods of time. PMID:27231016

  1. 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

  2. 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

  3. 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

  4. 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

  5. WinBEST-KIT: Windows-based biochemical reaction simulator for metabolic pathways.

    PubMed

    Sekiguchi, Tatsuya; Okamoto, Masahiro

    2006-06-01

    We have implemented an efficient, user-friendly biochemical reaction simulator called Web-based BEST-KIT (Biochemical Engineering System analyzing Tool-KIT) for analyzing large-scale nonlinear networks such as metabolic pathways. Users can easily design and analyze an arbitrary reaction scheme through the Internet and an efficient graphical user interface without considering the mathematical equations. The reaction scheme can include several reaction types, which are represented by both the mass action law (mass balance) and approximated velocity functions of enzyme kinetics at steady state, such as Michaelis-Menten, Hill cooperative, Competitive inhibition. However, since all modules in Web-based BEST-KIT have been developed in Java applet style, users cannot optionally make use of original mathematical equations in addition to the prepared equations. In the present study, we have developed a new version of BEST-KIT (for Microsoft Windows called WinBEST-KIT) to allow users to define original mathematical equations and to customize these equations very easily as user-defined reaction symbols. The following powerful system-analytical methods are prepared for system analysis: time-course calculation, parameter scanning, estimation of the values of unknown kinetic parameters based on experimentally observed time-course data of reactants, dynamic response of reactants against virtual external perturbations, and real-time simulation (Virtual Dry Lab). PMID:16960966

  6. Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway.

    PubMed

    Hicks, Katherine A; Ealick, Steven E

    2016-06-01

    HpxW from the ubiquitous pathogen Klebsiella pneumoniae is involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed. PMID:27303801

  7. 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, José; 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

  8. 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, β-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is

  9. 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

  10. 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

  11. 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

    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.

  12. Pathway Analyses Identify Novel Variants in the WNT Signaling Pathway Associated with Tuberculosis in Chinese Population

    PubMed Central

    Hu, Xuejiao; Zhou, Juan; Chen, Xuerong; Zhou, Yanhong; Song, Xingbo; Cai, Bei; Zhang, Jingya; Lu, Xiaojun; Ying, Binwu

    2016-01-01

    Tuberculosis remains a global public health problem, and its immunopathogenesis is still poorly understood. In this study, 25 single nucleotide polymorphisms (SNPs) in the WNT pathway were evaluated in relation to tuberculosis risk in a Chinese Han discovery set, and 6 candidate susceptible SNPs were further validated in a Chinese Tibetan cohort. Luciferase reporter assay, RT-qPCR and Western blot were used to assess the functionality of the important WNT polymorphisms. Five polymorphisms were associated with tuberculosis susceptibility after Bonferroni correction: SFRP1 rs4736958, CTNNB1 rs9859392, rs9870255 and rs3864004 showed decreased tuberculosis risk; SFRP1 rs7832767 was related to an increased risk (OR = 1.81, 95% CI = 1.30–2.52, p = 0.010). Patients with TT genotype of rs4736958 and rs7832767 correlated with higher CRP concentrations (p = 0.003, <0.001, respectively). Functional assays revealed that mutant alleles of rs9859392 (G), rs9870255 (C) and rs3864004 (A) were associated with significantly decreased transcriptional activity, lower CTNNB1 mRNA expression and p-β-catenin level, which were consistent with their effects of decreasing TB risk. Our results provide evidences that WNT pathway polymorphisms influence tuberculosis susceptibility and host immune response to Mycobacterium tuberculosis, suggesting that these variations may serve as novel markers for identifying the risk of developing tuberculosis. PMID:27334567

  13. 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

  14. 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

  15. Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

    PubMed Central

    Larkin, Angelyn; Chang, Michelle M.; Whitworth, Garrett E.; Imperiali, Barbara

    2013-01-01

    Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine (Dol-P-GlcNAc), which contrasts with the polyprenyl-diphosphate intermediates that feature in both eukaryotes and bacteria. Intriguingly, AglK exhibits high sequence homology to dolichyl-phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked Dol-P-glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide the first biochemical evidence revealing that despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life. PMID:23624439

  16. 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

  17. 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

  18. Biochemical pathway modeling tools for drug target detection in cancer and other complex diseases.

    PubMed

    Marin-Sanguino, Alberto; Gupta, Shailendra K; Voit, Eberhard O; Vera, Julio

    2011-01-01

    In the near future, computational tools and methods based on the mathematical modeling of biomedically relevant networks and pathways will be necessary for the design of therapeutic strategies that fight complex multifactorial diseases. Beyond the use of pharmacokinetic and pharmacodynamic approaches, we propose here the use of dynamic modeling as a tool for describing and analyzing the structure and responses of signaling, genetic and metabolic networks involved in such diseases. Specifically, we discuss the design and construction of meaningful models of biochemical networks, as well as tools, concepts, and strategies for using these models in the search of potential drug targets. We describe three different families of computational tools: predictive model simulations as tools for designing optimal drug profiles and doses; sensitivity analysis as a method to detect key interactions that affect critical outcomes and other characteristics of the network; and other tools integrating mathematical modeling with advanced computation and optimization for detecting potential drug targets. Furthermore, we show how potential drug targets detected with these approaches can be used in a computer-aided context to design or select new drug molecules. All concepts are illustrated with simplified examples and with actual case studies extracted from the recent literature. PMID:21187230

  19. Pathways-Driven Sparse Regression Identifies Pathways and Genes Associated with High-Density Lipoprotein Cholesterol in Two Asian Cohorts

    PubMed Central

    Silver, Matt; Chen, Peng; Li, Ruoying; Cheng, Ching-Yu; Wong, Tien-Yin; Tai, E-Shyong; Teo, Yik-Ying; Montana, Giovanni

    2013-01-01

    Standard approaches to data analysis in genome-wide association studies (GWAS) ignore any potential functional relationships between gene variants. In contrast gene pathways analysis uses prior information on functional structure within the genome to identify pathways associated with a trait of interest. In a second step, important single nucleotide polymorphisms (SNPs) or genes may be identified within associated pathways. The pathways approach is motivated by the fact that genes do not act alone, but instead have effects that are likely to be mediated through their interaction in gene pathways. Where this is the case, pathways approaches may reveal aspects of a trait's genetic architecture that would otherwise be missed when considering SNPs in isolation. Most pathways methods begin by testing SNPs one at a time, and so fail to capitalise on the potential advantages inherent in a multi-SNP, joint modelling approach. Here, we describe a dual-level, sparse regression model for the simultaneous identification of pathways and genes associated with a quantitative trait. Our method takes account of various factors specific to the joint modelling of pathways with genome-wide data, including widespread correlation between genetic predictors, and the fact that variants may overlap multiple pathways. We use a resampling strategy that exploits finite sample variability to provide robust rankings for pathways and genes. We test our method through simulation, and use it to perform pathways-driven gene selection in a search for pathways and genes associated with variation in serum high-density lipoprotein cholesterol levels in two separate GWAS cohorts of Asian adults. By comparing results from both cohorts we identify a number of candidate pathways including those associated with cardiomyopathy, and T cell receptor and PPAR signalling. Highlighted genes include those associated with the L-type calcium channel, adenylate cyclase, integrin, laminin, MAPK signalling and immune

  20. Concordant Signaling Pathways Produced by Pesticide Exposure in Mice Correspond to Pathways Identified in Human Parkinson's Disease

    PubMed Central

    Gollamudi, Seema; Johri, Ashu; Calingasan, Noel Y.; Yang, Lichuan; Elemento, Olivier; Beal, M. Flint

    2012-01-01

    Parkinson's disease (PD) is a neurodegenerative disease in which the etiology of 90 percent of the patients is unknown. Pesticide exposure is a major risk factor for PD, and paraquat (PQ), pyridaben (PY) and maneb (MN) are amongst the most widely used pesticides. We studied mRNA expression using transcriptome sequencing (RNA-Seq) in the ventral midbrain (VMB) and striatum (STR) of PQ, PY and paraquat+maneb (MNPQ) treated mice, followed by pathway analysis. We found concordance of signaling pathways between the three pesticide models in both the VMB and STR as well as concordance in these two brain areas. The concordant signaling pathways with relevance to PD pathogenesis were e.g. axonal guidance signaling, Wnt/β-catenin signaling, as well as pathways not previously linked to PD, e.g. basal cell carcinoma, human embryonic stem cell pluripotency and role of macrophages, fibroblasts and endothelial cells in rheumatoid arthritis. Human PD pathways previously identified by expression analysis, concordant with VMB pathways identified in our study were axonal guidance signaling, Wnt/β-catenin signaling, IL-6 signaling, ephrin receptor signaling, TGF-β signaling, PPAR signaling and G-protein coupled receptor signaling. Human PD pathways concordant with the STR pathways in our study were Wnt/β-catenin signaling, axonal guidance signaling and G-protein coupled receptor signaling. Peroxisome proliferator activated receptor delta (Ppard) and G-Protein Coupled Receptors (GPCRs) were common genes in VMB and STR identified by network analysis. In conclusion, the pesticides PQ, PY and MNPQ elicit common signaling pathways in the VMB and STR in mice, which are concordant with known signaling pathways identified in human PD, suggesting that these pathways contribute to the pathogenesis of idiopathic PD. The analysis of these networks and pathways may therefore lead to improved understanding of disease pathogenesis, and potential novel therapeutic targets. PMID:22563483

  1. Site-specific isotope fractionation in the characterization of biochemical mechanisms. The glycolytic pathway.

    PubMed

    Zhang, B L; Yunianta; Martin, M L

    1995-07-01

    For a given biochemical transformation, such as the fermentation reaction, the redistribution coefficients, which relate the natural site-specific isotope contents in end products to those of their precursors, are a source of mechanistic information. These coefficients characterize the traceability of specific hydrogens in the products (ethanol and water) to their parent hydrogens in the starting materials (glucose and water). In conditions of complete transformation, they also enable intermolecular exchanges with the water medium to be estimated. Thus it is directly confirmed that hydrogens 1, 2, 6, and 6' of glucose are strongly connected to the methyl site I of ethanol obtained by fermentation by Saccharomyces cerevisiae. However, whereas hydrogens 6 and 6' are transferred to a great extent, transfer is only partial for hydrogen 2, and it is even less for hydrogen 1. Because the two moieties of glucose corresponding to carbons 1-2-3 and 4-5-6 are scrambled by the aldolase and triosephosphate isomerase reactions, additional exchange of hydrogens at positions 1 and 2 must have occurred before these steps. The value of the coefficient that relates site 2 of glucose to site I of ethanol in particular can be used to quantify the contribution of intermolecular exchange occurring in the course of the transfer from site 2 of glucose 6-phosphate to site 1 of fructose 6-phosphate mediated by phosphoglucoisomerase. The average hydrogen isotope effects associated with the transfer of hydrogen from the water pool to the methyl or methylene site of ethanol are estimated. In contrast to conventional experiments carried out in strongly deuterium-enriched media where metabolic switching may occur, the NMR investigation of site-specific natural isotope fractionation, which operates at tracer isotopic abundance, faithfully describes the unperturbed metabolic pathways. PMID:7608163

  2. Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria.

    PubMed

    Barth, Kenneth R; Isabella, Vincent M; Clark, Virginia L

    2009-12-01

    Since Neisseria gonorrhoeae and Neisseria meningitidis are obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensal Neisseria genome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. Only Neisseria mucosa contained a nitrate reductase (Nar), and only Neisseria lactamica, Neisseria cinerea, Neisseria subflava, Neisseria flavescens and Neisseria sicca contained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested except N. mucosa had a two- to fourfold lower nitrite reductase activity than N. gonorrhoeae, while N. meningitidis and most of the commensal Neisseria species had a two- to fourfold higher nitric oxide (NO) reductase activity. For N. meningitidis and most of the commensal Neisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared with N. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that of N. gonorrhoeae. The greatest difference between the Neisseria species was the lack of a functional Nos system in the pathogenic species N. gonorrhoeae and N. meningitidis. PMID:19762442

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

    PubMed

    Phenix, Hilary; Perkins, Theodore; Kærn, 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

  4. Identifiability and inference of pathway motifs by epistasis analysis

    NASA Astrophysics Data System (ADS)

    Phenix, Hilary; Perkins, Theodore; Kærn, 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.

  5. Training Signaling Pathway Maps to Biochemical Data with Constrained Fuzzy Logic: Quantitative Analysis of Liver Cell Responses to Inflammatory Stimuli

    PubMed Central

    Morris, Melody K.; Saez-Rodriguez, Julio; Clarke, David C.; Sorger, Peter K.; Lauffenburger, Douglas A.

    2011-01-01

    Predictive understanding of cell signaling network operation based on general prior knowledge but consistent with empirical data in a specific environmental context is a current challenge in computational biology. Recent work has demonstrated that Boolean logic can be used to create context-specific network models by training proteomic pathway maps to dedicated biochemical data; however, the Boolean formalism is restricted to characterizing protein species as either fully active or inactive. To advance beyond this limitation, we propose a novel form of fuzzy logic sufficiently flexible to model quantitative data but also sufficiently simple to efficiently construct models by training pathway maps on dedicated experimental measurements. Our new approach, termed constrained fuzzy logic (cFL), converts a prior knowledge network (obtained from literature or interactome databases) into a computable model that describes graded values of protein activation across multiple pathways. We train a cFL-converted network to experimental data describing hepatocytic protein activation by inflammatory cytokines and demonstrate the application of the resultant trained models for three important purposes: (a) generating experimentally testable biological hypotheses concerning pathway crosstalk, (b) establishing capability for quantitative prediction of protein activity, and (c) prediction and understanding of the cytokine release phenotypic response. Our methodology systematically and quantitatively trains a protein pathway map summarizing curated literature to context-specific biochemical data. This process generates a computable model yielding successful prediction of new test data and offering biological insight into complex datasets that are difficult to fully analyze by intuition alone. PMID:21408212

  6. CyanoPhyChe: A Database for Physico-Chemical Properties, Structure and Biochemical Pathway Information of Cyanobacterial Proteins

    PubMed Central

    Arun, P. V. Parvati Sai; Bakku, Ranjith Kumar; Subhashini, Mranu; Singh, Pankaj; Prabhu, N. Prakash; Suzuki, Iwane; Prakash, Jogadhenu S. S.

    2012-01-01

    CyanoPhyChe is a user friendly database that one can browse through for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins. We downloaded all the protein sequences from the cyanobacterial genome database for calculating the physico-chemical properties, such as molecular weight, net charge of protein, isoelectric point, molar extinction coefficient, canonical variable for solubility, grand average hydropathy, aliphatic index, and number of charged residues. Based on the physico-chemical properties, we provide the polarity, structural stability and probability of a protein entering in to an inclusion body (PEPIB). We used the data generated on physico-chemical properties, structure and biochemical pathway information of all cyanobacterial proteins to construct CyanoPhyChe. The data can be used for optimizing methods of expression and characterization of cyanobacterial proteins. Moreover, the ‘Search’ and data export options provided will be useful for proteome analysis. Secondary structure was predicted for all the cyanobacterial proteins using PSIPRED tool and the data generated is made accessible to researchers working on cyanobacteria. In addition, external links are provided to biological databases such as PDB and KEGG for molecular structure and biochemical pathway information, respectively. External links are also provided to different cyanobacterial databases. CyanoPhyChe can be accessed from the following URL: http://bif.uohyd.ac.in/cpc. PMID:23185330

  7. Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus Neurospora crassa Ergothioneine Biosynthetic Pathway

    PubMed Central

    2015-01-01

    Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C–S bond formation and a PLP-mediated C–S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C–S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible. PMID:25275953

  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. 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.

  10. 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.

  11. Characterization of a novel oxyfluorfen-degrading bacterial strain Chryseobacterium aquifrigidense and its biochemical degradation pathway.

    PubMed

    Zhao, Huanhuan; Xu, Jun; Dong, Fengshou; Liu, Xingang; Wu, Yanbing; Wu, Xiaohu; Zheng, Yongquan

    2016-08-01

    Persistent use of the diphenyl ether herbicides oxyfluorfen may seriously increase the health risks and ecological safety problems. A newly bacterium R-21 isolated from active soil was able to degrade and utilize oxyfluorfen as the sole carbon source. R-21 was identified as Chryseobacterium aquifrigidense by morphology, physiobiochemical characteristics, and genetic analysis. Under the optimum cultural conditions (pH 6.9, temperature 33.4 °C, and inoculum size 0.2 g L(-1)), R-21 could degrade 92.1 % of oxyfluorfen at 50 mg L(-1) within 5 days. During oxyfluorfen degradation, six metabolites were detected and identified by atmospheric pressure gas chromatography coupled to quadrupole-time of flight mass spectrometry and ultra-performance liquid chromatography coupled to quadrupole-time of flight mass spectrometry, and a plausible degradation pathway was deduced. Strain R-21 is a promising potential in bioremediation of oxyfluorfen-contaminated environments. PMID:27079576

  12. 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

  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

  14. Neuropathological and biochemical criteria to identify acquired Creutzfeldt-Jakob disease among presumed sporadic cases.

    PubMed

    Kobayashi, Atsushi; Parchi, Piero; Yamada, Masahito; Mohri, Shirou; Kitamoto, Tetsuyuki

    2016-06-01

    As an experimental model of acquired Creutzfeldt-Jakob disease (CJD), we performed transmission studies of sporadic CJD using knock-in mice expressing human prion protein (PrP). In this model, the inoculation of the sporadic CJD strain V2 into animals homozygous for methionine at polymorphic codon 129 (129 M/M) of the PRNP gene produced quite distinctive neuropathological and biochemical features, that is, widespread kuru plaques and intermediate type abnormal PrP (PrP(Sc) ). Interestingly, this distinctive combination of molecular and pathological features has been, to date, observed in acquired CJD but not in sporadic CJD. Assuming that these distinctive phenotypic traits are specific for acquired CJD, we revisited the literature and found two cases showing widespread kuru plaques despite the 129 M/M genotype, in a neurosurgeon and in a patient with a medical history of neurosurgery without dura mater grafting. By Western blot analysis of brain homogenates, we revealed the intermediate type of PrP(Sc) in both cases. Furthermore, transmission properties of brain extracts from these two cases were indistinguishable from those of a subgroup of dura mater graft-associated iatrogenic CJD caused by infection with the sporadic CJD strain V2. These data strongly suggest that the two atypical CJD cases, previously thought to represent sporadic CJD, very likely acquired the disease through exposure to prion-contaminated brain tissues. Thus, we propose that the distinctive combination of 129 M/M genotype, kuru plaques, and intermediate type PrP(Sc) , represents a reliable criterion for the identification of acquired CJD cases among presumed sporadic cases. PMID:26669818

  15. A cross-study gene set enrichment analysis identifies critical pathways in endometriosis

    PubMed Central

    Zhao, Hongbo; Wang, Qishan; Bai, Chunyan; He, Kan; Pan, Yuchun

    2009-01-01

    Background Endometriosis is an enigmatic disease. Gene expression profiling of endometriosis has been used in several studies, but few studies went further to classify subtypes of endometriosis based on expression patterns and to identify possible pathways involved in endometriosis. Some of the observed pathways are more inconsistent between the studies, and these candidate pathways presumably only represent a fraction of the pathways involved in endometriosis. Methods We applied a standardised microarray preprocessing and gene set enrichment analysis to six independent studies, and demonstrated increased concordance between these gene datasets. Results We find 16 up-regulated and 19 down-regulated pathways common in ovarian endometriosis data sets, 22 up-regulated and one down-regulated pathway common in peritoneal endometriosis data sets. Among them, 12 up-regulated and 1 down-regulated were found consistent between ovarian and peritoneal endometriosis. The main canonical pathways identified are related to immunological and inflammatory disease. Early secretory phase has the most over-represented pathways in the three uterine cycle phases. There are no overlapping significant pathways between the dataset from human endometrial endothelial cells and the datasets from ovarian endometriosis which used whole tissues. Conclusion The study of complex diseases through pathway analysis is able to highlight genes weakly connected to the phenotype which may be difficult to detect by using classical univariate statistics. By standardised microarray preprocessing and GSEA, we have increased the concordance in identifying many biological mechanisms involved in endometriosis. The identified gene pathways will shed light on the understanding of endometriosis and promote the development of novel therapies. PMID:19735579

  16. 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.

  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. 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

  19. A search engine to identify pathway genes from expression data on multiple organisms

    PubMed Central

    Chen, Chunnuan; Weirauch, Matthew T; Powell, Corey C; Zambon, Alexander C; Stuart, Joshua M

    2007-01-01

    Background The completion of several genome projects showed that most genes have not yet been characterized, especially in multicellular organisms. Although most genes have unknown functions, a large collection of data is available describing their transcriptional activities under many different experimental conditions. In many cases, the coregulatation of a set of genes across a set of conditions can be used to infer roles for genes of unknown function. Results We developed a search engine, the Multiple-Species Gene Recommender (MSGR), which scans gene expression datasets from multiple organisms to identify genes that participate in a genetic pathway. The MSGR takes a query consisting of a list of genes that function together in a genetic pathway from one of six organisms: Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Arabidopsis thaliana, and Helicobacter pylori. Using a probabilistic method to merge searches, the MSGR identifies genes that are significantly coregulated with the query genes in one or more of those organisms. The MSGR achieves its highest accuracy for many human pathways when searches are combined across species. We describe specific examples in which new genes were identified to be involved in a neuromuscular signaling pathway and a cell-adhesion pathway. Conclusion The search engine can scan large collections of gene expression data for new genes that are significantly coregulated with a pathway of interest. By integrating searches across organisms, the MSGR can identify pathway members whose coregulation is either ancient or newly evolved. PMID:17477880

  20. Influence maximization in time bounded network identifies transcription factors regulating perturbed pathways

    PubMed Central

    Jo, Kyuri; Jung, Inuk; Moon, Ji Hwan; Kim, Sun

    2016-01-01

    Motivation: To understand the dynamic nature of the biological process, it is crucial to identify perturbed pathways in an altered environment and also to infer regulators that trigger the response. Current time-series analysis methods, however, are not powerful enough to identify perturbed pathways and regulators simultaneously. Widely used methods include methods to determine gene sets such as differentially expressed genes or gene clusters and these genes sets need to be further interpreted in terms of biological pathways using other tools. Most pathway analysis methods are not designed for time series data and they do not consider gene-gene influence on the time dimension. Results: In this article, we propose a novel time-series analysis method TimeTP for determining transcription factors (TFs) regulating pathway perturbation, which narrows the focus to perturbed sub-pathways and utilizes the gene regulatory network and protein–protein interaction network to locate TFs triggering the perturbation. TimeTP first identifies perturbed sub-pathways that propagate the expression changes along the time. Starting points of the perturbed sub-pathways are mapped into the network and the most influential TFs are determined by influence maximization technique. The analysis result is visually summarized in TF-Pathway map in time clock. TimeTP was applied to PIK3CA knock-in dataset and found significant sub-pathways and their regulators relevant to the PIP3 signaling pathway. Availability and Implementation: TimeTP is implemented in Python and available at http://biohealth.snu.ac.kr/software/TimeTP/. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: sunkim.bioinfo@snu.ac.kr PMID:27307609

  1. 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-α subunit and stimulated intracellular cAMP. At the present, many researchers defined agonist or antagonist activity of potential GPCRs drugs employing [35S]GTPγS 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

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

    DOEpatents

    Holzrichter, John F.; Siekhaus, Wigbert J.

    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.

  3. 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.

  4. Biochemical pathways that regulate acetyltransferase and deacetylase activity in mammalian cells

    PubMed Central

    Mellert, Hestia S.; McMahon, Steven B.

    2009-01-01

    Protein phosphorylation is dynamically regulated in eukaryotic cells via modulation of the enzymatic activity of kinases and phosphatases. Like phosphorylation, acetylation has emerged as a critical regulatory protein modification that is dynamically altered in response to diverse cellular cues. Moreover, acetyltransferases and deacetylases are tightly linked to cellular signaling pathways. Recent studies provide clues about the mechanisms utilized to regulate acetyltransferases and deacetylases. The therapeutic value of deacetylase inhibitors suggests that understanding acetylation pathways will directly impact our ability to rationally target these enzymes in patients. Recently discovered mechanisms which directly regulate the catalytic activity of acetyltransferases and deacetylases provide exciting new insights about these enzymes. PMID:19819149

  5. Elucidation of the biochemical pathway of 2-phenylethanol from shikimic acid using isolated protoplasts of rose flowers.

    PubMed

    Yang, Ziyin; Sakai, Miwa; Sayama, Hironori; Shimeno, Taku; Yamaguchi, Koji; Watanabe, Naoharu

    2009-05-15

    The isolated protoplasts of rose flowers were used to investigate the metabolic pathway in rose flower leading from shikimic acid or L-phenylalanine (L-Phe) to 2-phenylethanol (2PE), a dominant volatile compound in hybrid roses such as Rosa damascena Mill., R. 'Hoh-Jun', and R. 'Yves Piaget'. Deuterium-labeled L-Phe ([2H8]L-Phe) was supplied to the protoplasts isolated from R. 'Yves Piaget' petals. The volatile end products ([2Hn]-2PE, n=6-8) and their related intermediates ([2Hn]phenylacetaldehyde, n=6-8) were detected in the protoplasts by gas chromatography-mass spectrometry (GC-MS). In addition, we chemically synthesized [2,3,4,5,6-13C5]shikimic acid, a new stable isotopomer, to investigate the formation of 2PE from shikimic acid by GC-MS and nuclear magnetic resonance. We proposed the hypothetical biochemical pathway of 2PE from shikimic acid via chorismic acid, L-Phe, and phenylacetaldehyde. This protoplast system facilitates findings of metabolic intermediates and simplifies the complex branching biosynthetic pathways of floral scents to distinct individual events. PMID:19097671

  6. 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

  7. 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…

  8. Biochemical pathways of breath ammonia (NH3) generation in patients with end-stage renal disease undergoing hemodialysis.

    PubMed

    Chen, W; Laiho, S; Vaittinen, O; Halonen, L; Ortiz, F; Forsblom, C; Groop, P-H; Lehto, M; Metsälä, M

    2016-01-01

    Breath ammonia (NH3) has been proposed as a potential biomarker in monitoring hemodialysis (HD) adequacy, since a strong correlation between blood urea and mouth-exhaled breath NH3 has been observed in patients with end-stage renal disease (ESRD) undergoing HD. However, the biochemical pathways for breath NH3 generation from blood urea have not been demonstrated. In this study, we show a strong correlation (r s  =  0.77, p  <  0.001) between blood and salivary urea, indicating that salivary urea levels reflect blood urea levels. Salivary urea is in turn strongly correlated to salivary ammonia ([Formula: see text] + NH3) in most of the patients. This confirms that the hydrolysis of urea by urease generates ammonia in the oral cavity. A further strong correlation between salivary ammonia and breath NH3 indicates that salivary ammonia evaporates into gas phase and turns to breath NH3. Therefore, blood urea is a major biochemical source of breath NH3. Since breath NH3 is generated predominantly in the oral cavity, the levels of breath NH3 are influenced significantly by the patient's oral condition including urease activity and salivary pH. Our results agree with previous studies that have shown a connection between salivary urea and breath NH3. PMID:27516572

  9. The biochemical pathway for the breakdown of methyl cyanide (acetonitrile) in bacteria.

    PubMed Central

    Firmin, J L; Gray, D O

    1976-01-01

    [2-14C]Methyl cyanide (acetonitrile) is metabolized to citrate, succinate, fumarate, malate, glutamate, pyrrolidonecarboxylic acid and aspartate. Non-radioactive acetamide and acetate compete with 14C from methyl cyanide, and [2-14C]acetate and [2-14C]methyl cyanide are metabolized at similar rates, giving identical products. This evidence, combined with the inhibitory effect of fluoroacetate and arsenite on methyl cyanide metabolism, indicates that the pathway is: methyl cyanide leads to acetamide leads to acetate leads to tricarboxylic acid-cycle intermediates. The pathway was investigated in a species of Pseudomonas (group III; N.C.I.B. 10477), but comparison of labelling patterns suggests that it also exists in several higher plants. PMID:985423

  10. Cartography of Pathway Signal Perturbations Identifies Distinct Molecular Pathomechanisms in Malignant and Chronic Lung Diseases

    PubMed Central

    Arakelyan, Arsen; Nersisyan, Lilit; Petrek, Martin; Löffler-Wirth, Henry; Binder, Hans

    2016-01-01

    Lung diseases are described by a wide variety of developmental mechanisms and clinical manifestations. Accurate classification and diagnosis of lung diseases are the bases for development of effective treatments. While extensive studies are conducted toward characterization of various lung diseases at molecular level, no systematic approach has been developed so far. Here we have applied a methodology for pathway-centered mining of high throughput gene expression data to describe a wide range of lung diseases in the light of shared and specific pathway activity profiles. We have applied an algorithm combining a Pathway Signal Flow (PSF) algorithm for estimation of pathway activity deregulation states in lung diseases and malignancies, and a Self Organizing Maps algorithm for classification and clustering of the pathway activity profiles. The analysis results allowed clearly distinguish between cancer and non-cancer lung diseases. Lung cancers were characterized by pathways implicated in cell proliferation, metabolism, while non-malignant lung diseases were characterized by deregulations in pathways involved in immune/inflammatory response and fibrotic tissue remodeling. In contrast to lung malignancies, chronic lung diseases had relatively heterogeneous pathway deregulation profiles. We identified three groups of interstitial lung diseases and showed that the development of characteristic pathological processes, such as fibrosis, can be initiated by deregulations in different signaling pathways. In conclusion, this paper describes the pathobiology of lung diseases from systems viewpoint using pathway centered high-dimensional data mining approach. Our results contribute largely to current understanding of pathological events in lung cancers and non-malignant lung diseases. Moreover, this paper provides new insight into molecular mechanisms of a number of interstitial lung diseases that have been studied to a lesser extent. PMID:27200087

  11. Cartography of Pathway Signal Perturbations Identifies Distinct Molecular Pathomechanisms in Malignant and Chronic Lung Diseases.

    PubMed

    Arakelyan, Arsen; Nersisyan, Lilit; Petrek, Martin; Löffler-Wirth, Henry; Binder, Hans

    2016-01-01

    Lung diseases are described by a wide variety of developmental mechanisms and clinical manifestations. Accurate classification and diagnosis of lung diseases are the bases for development of effective treatments. While extensive studies are conducted toward characterization of various lung diseases at molecular level, no systematic approach has been developed so far. Here we have applied a methodology for pathway-centered mining of high throughput gene expression data to describe a wide range of lung diseases in the light of shared and specific pathway activity profiles. We have applied an algorithm combining a Pathway Signal Flow (PSF) algorithm for estimation of pathway activity deregulation states in lung diseases and malignancies, and a Self Organizing Maps algorithm for classification and clustering of the pathway activity profiles. The analysis results allowed clearly distinguish between cancer and non-cancer lung diseases. Lung cancers were characterized by pathways implicated in cell proliferation, metabolism, while non-malignant lung diseases were characterized by deregulations in pathways involved in immune/inflammatory response and fibrotic tissue remodeling. In contrast to lung malignancies, chronic lung diseases had relatively heterogeneous pathway deregulation profiles. We identified three groups of interstitial lung diseases and showed that the development of characteristic pathological processes, such as fibrosis, can be initiated by deregulations in different signaling pathways. In conclusion, this paper describes the pathobiology of lung diseases from systems viewpoint using pathway centered high-dimensional data mining approach. Our results contribute largely to current understanding of pathological events in lung cancers and non-malignant lung diseases. Moreover, this paper provides new insight into molecular mechanisms of a number of interstitial lung diseases that have been studied to a lesser extent. PMID:27200087

  12. 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

  13. 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.

  14. Using mechanistic Bayesian networks to identify downstream targets of the Sonic Hedgehog pathway

    PubMed Central

    2009-01-01

    Background The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge. Results We introduce a new general-purpose analytic method called Mechanistic Bayesian Networks (MBNs) that allows for the integration of gene expression data and known constraints within a signal or regulatory pathway to predict new downstream pathway targets. The MBN framework is implemented in an open-source Bayesian network learning package, the Python Environment for Bayesian Learning (PEBL). We demonstrate how MBNs can be used by modeling the early steps of the sonic hedgehog pathway using gene expression data from different developmental stages and genetic backgrounds in mouse. Using the MBN approach we are able to automatically identify many of the known downstream targets of the hedgehog pathway such as Gas1 and Gli1, along with a short list of likely targets such as Mig12. Conclusions The MBN approach shown here can easily be extended to other pathways and data types to yield a more mechanistic framework for learning genetic regulatory models. PMID:20021670

  15. 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.

  16. 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

  17. Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6.

    PubMed

    Pereira, Jose Henrique; Heins, Richard A; Gall, Daniel L; McAndrew, Ryan P; Deng, Kai; Holland, Keefe C; Donohue, Timothy J; Noguera, Daniel R; Simmons, Blake A; Sale, Kenneth L; Ralph, John; Adams, Paul D

    2016-05-01

    There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50-70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts. PMID:26940872

  18. Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6*

    PubMed Central

    Pereira, Jose Henrique; Heins, Richard A.; Gall, Daniel L.; McAndrew, Ryan P.; Deng, Kai; Holland, Keefe C.; Donohue, Timothy J.; Noguera, Daniel R.; Simmons, Blake A.; Sale, Kenneth L.; Ralph, John; Adams, Paul D.

    2016-01-01

    There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts. PMID:26940872

  19. Gene interaction enrichment and network analysis to identify dysregulated pathways and their interactions in complex diseases

    PubMed Central

    2012-01-01

    Background The molecular behavior of biological systems can be described in terms of three fundamental components: (i) the physical entities, (ii) the interactions among these entities, and (iii) the dynamics of these entities and interactions. The mechanisms that drive complex disease can be productively viewed in the context of the perturbations of these components. One challenge in this regard is to identify the pathways altered in specific diseases. To address this challenge, Gene Set Enrichment Analysis (GSEA) and others have been developed, which focus on alterations of individual properties of the entities (such as gene expression). However, the dynamics of the interactions with respect to disease have been less well studied (i.e., properties of components ii and iii). Results Here, we present a novel method called Gene Interaction Enrichment and Network Analysis (GIENA) to identify dysregulated gene interactions, i.e., pairs of genes whose relationships differ between disease and control. Four functions are defined to model the biologically relevant gene interactions of cooperation (sum of mRNA expression), competition (difference between mRNA expression), redundancy (maximum of expression), or dependency (minimum of expression) among the expression levels. The proposed framework identifies dysregulated interactions and pathways enriched in dysregulated interactions; points out interactions that are perturbed across pathways; and moreover, based on the biological annotation of each type of dysregulated interaction gives clues about the regulatory logic governing the systems level perturbation. We demonstrated the potential of GIENA using published datasets related to cancer. Conclusions We showed that GIENA identifies dysregulated pathways that are missed by traditional enrichment methods based on the individual gene properties and that use of traditional methods combined with GIENA provides coverage of the largest number of relevant pathways. In addition

  20. An Integrated Human/Murine Transcriptome and Pathway Approach To Identify Prenatal Treatments For Down Syndrome.

    PubMed

    Guedj, Faycal; Pennings, Jeroen LA; Massingham, Lauren J; Wick, Heather C; Siegel, Ashley E; Tantravahi, Umadevi; Bianchi, Diana W

    2016-01-01

    Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls, and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn, and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice. PMID:27586445

  1. An Integrated Human/Murine Transcriptome and Pathway Approach To Identify Prenatal Treatments For Down Syndrome

    PubMed Central

    Guedj, Faycal; Pennings, Jeroen LA; Massingham, Lauren J.; Wick, Heather C.; Siegel, Ashley E.; Tantravahi, Umadevi; Bianchi, Diana W.

    2016-01-01

    Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls, and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn, and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice. PMID:27586445

  2. Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

    PubMed Central

    Wang, Hongchao; Yang, Bo; Hao, Guangfei; Feng, Yun; Chen, Haiqin; Feng, Lu; Zhao, Jianxin; Zhang, Hao; Chen, Yong Q.; Wang, Lei

    2011-01-01

    We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH4) in the lipid-producing fungus Mortierella alpina. The BH4 cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H2-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H2-NTP to 6-pyruvoyltetrahydropterin (PPH4), and sepiapterin reductase (SR) for the conversion of PPH4 to BH4, were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH4 in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH4 and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH4 biosynthesis pathway in a fungus. PMID:21852350

  3. Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways.

    PubMed

    Hudson, André O; Harkness, Taylor C M; Savka, Michael A

    2016-01-01

    Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very common in biochemistry, genetics and many other disciplines. A comprehensive overview of the technique to supplement the teaching of biochemical pathways pertaining to amino acids, peptidoglycan and the bacterial stringent response is reported in this manuscript. Two cDNAs from the model plant organism Arabidopsis thaliana that are involved in the metabolism of lysine (L,L-diaminopimelate aminotransferase (dapL) and tyrosine aminotransferase (tyrB) involved in the metabolism of tyrosine and phenylalanine are highlighted. In addition, the bacterial peptidoglycan anabolic pathway is highlighted through the analysis of the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate ligase (murE) gene from the bacterium Verrucomicrobium spinosum involved in the cross-linking of peptidoglycan. The bacterial stringent response is also reported through the analysis of the rsh (relA/spoT homolog) bifunctional gene responsible for a hyper-mucoid phenotype in the bacterium Novosphingobium sp. Four examples of FCA are presented. The video will focus on three of them, namely lysine, peptidoglycan and the stringent response. PMID:27403640

  4. An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: A biochemical and molecular perspective*

    PubMed Central

    Cheng, C. Yan; Mruk, Dolores D.

    2009-01-01

    During spermatogenesis, fully developed spermatids (i.e., spermatozoa) at the luminal edge of the seminiferous epithelium undergo ‘spermiation’ at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood-testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to ‘destabilize’ the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood-tissue barriers. PMID:19622063

  5. Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing.

    PubMed

    Drissen, Roy; Buza-Vidas, Natalija; Woll, Petter; Thongjuea, Supat; Gambardella, Adriana; Giustacchini, Alice; Mancini, Elena; Zriwil, Alya; Lutteropp, Michael; Grover, Amit; Mead, Adam; Sitnicka, Ewa; Jacobsen, Sten Eirik W; Nerlov, Claus

    2016-06-01

    According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)Flt3(hi)) and common myeloid progenitors (CMPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)CD41(hi)) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre-granulocyte-macrophage progenitor (pre-GM) (Lin(-)Sca-1(-)c-Kit(+)CD41(-)FcγRII/III(-)CD150(-)CD105(-)). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential. PMID:27043410

  6. 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.

  7. Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons.

    PubMed

    Weretilnyk, E A; Bednarek, S; McCue, K F; Rhodes, D; Hanson, A D

    1989-06-01

    Members of the Chenopodiaceae can accumulate high levels (>100 μmol·(g DW)(-1)) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (choline→betaine aldehyde→ betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 μmol·(g DW)(-1) are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1-0.5 μmol·(g DW)(-1). Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the Mr of the BADH monomer is in all cases close to 63 000. These data indicate that the choline→betaine aldehyde→betaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of Mr 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea. PMID:24212901

  8. 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.

  9. Probabilistic approach to identify sensitive parameter distributions in multimedia pathway analysis.

    SciTech Connect

    Kamboj, S.; Gnanapragasam, E.; LePoire, D.; Biwer, B. M.; Cheng, J.; Arnish, J.; Yu, C.; Chen, S. Y.; Mo, T.; Abu-Eid, R.; Thaggard, M.; Environmental Assessment; NRC

    2002-01-01

    Sensitive parameter distributions were identified with the use of probabilistic analysis in the RESRAD computer code. RESRAD is a multimedia pathway analysis code designed to evaluate radiological exposures resulting from radiological contamination in soil. The dose distribution was obtained by using a set of default parameter distribution/values. Most of the variations in the output dose distribution could be attributed to uncertainty in a small set of input parameters that could be considered as sensitive parameter distributions. The identification of the sensitive parameters is a first step in the prioritization of future research and information gathering. When site-specific parameter distribution/values are available for an actual site, the same process should be used with these site-specific data. Regression analysis used to identify sensitive parameters indicated that the dominant pathways depended on the radionuclide and source configurations. However, two parameter distributions were sensitive for many radionuclides: the external shielding factor when external exposure was the dominant pathway and the plant transfer factor when plant ingestion was the dominant pathway. No single correlation or regression coefficient can be used alone to identify sensitive parameters in all the cases. The coefficients are useful guides, but they have to be used in conjunction with other aids, such as scatter plots, and should undergo further analysis.

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

    PubMed Central

    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

  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. 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...

  12. 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.

  13. Integrative analysis of genome-wide association studies and gene expression analysis identifies pathways associated with rheumatoid arthritis

    PubMed Central

    Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

    2016-01-01

    Rheumatoid arthritis (RA) is a complex and systematic autoimmune disease, which is usually influenced by both genetic and environmental factors. Pathway analyses based on a single data type such as microarray data or SNP data have successfully revealed some biology pathways associated with RA. However, we found that the pathway analysis based on a single data type only provide limited understanding about the pathogenesis of RA. Gene-disease association is usually caused by many ways, such as genotype, gene expression and so on. Therefore, the integrative analysis method combining multiple levels of evidence can more precisely and comprehensively identify the pathway associations. In this study, we performed a pathway analysis by integrating GWAS and gene expression analysis to detect the RA-related pathways. The integrative analysis identified 28 pathways associated with RA. Among these pathways, 18 pathways were also found by both GWAS and gene expression analysis, 7 pathways are novel RA-related pathways, such as B cell receptor signaling pathway, Toll-like receptor signaling pathway, Fc gamma R-mediated phagocytosis and so on. Compared with pathway analyses using only one type genomic data, we found integrative analysis can increase the power to identify the real associations and provided more stable and accurate results. We believe these results will contribute to perform future genetic studies in RA pathogenesis and may promote the development of new therapeutic strategies by targeting these pathways. PMID:26885899

  14. Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma

    PubMed Central

    Byrum, Stephanie D.; Larson, Signe K.; Avaritt, Nathan L.; Moreland, Linley E.; Mackintosh, Samuel G.; Cheung, Wang L.; Tackett, Alan J.

    2013-01-01

    Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression. PMID:23976835

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

    PubMed

    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

  16. 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

  17. Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

    PubMed Central

    Gimigliano, Anna; Mannini, Linda; Bianchi, Laura; Puglia, Michele; Deardorff, Matthew A.; Menga, Stefania; Krantz, Ian D; Musio, Antonio; Bini, Luca

    2012-01-01

    Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, SMC1A and SMC3. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the c-MYC gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS. PMID:23106691

  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. Simulated annealing based algorithm for identifying mutated driver pathways in cancer.

    PubMed

    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

  20. 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.

  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. Global Biochemical Profiling Identifies β-Hydroxypyruvate as a Potential Mediator of Type 2 Diabetes in Mice and Humans

    PubMed Central

    Zhang, Sheng; Wang, Songyan; Puhl, Matthew D.; Jiang, Xuntian; Hyrc, Krzysztof L.; Laciny, Erin; Wallendorf, Michael J.; Pappan, Kirk L.; Coyle, Joseph T.

    2015-01-01

    Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are incretins secreted by respective K and L enteroendocrine cells after eating and amplify glucose-stimulated insulin secretion (GSIS). This amplification has been termed the “incretin response.” To determine the role(s) of K cells for the incretin response and type 2 diabetes mellitus (T2DM), diphtheria toxin–expressing (DT) mice that specifically lack GIP-producing cells were backcrossed five to eight times onto the diabetogenic NONcNZO10/Ltj background. As in humans with T2DM, DT mice lacked an incretin response, although GLP-1 release was maintained. With high-fat (HF) feeding, DT mice remained lean but developed T2DM, whereas wild-type mice developed obesity but not diabetes. Metabolomics identified biochemicals reflecting impaired glucose handling, insulin resistance, and diabetes complications in prediabetic DT/HF mice. β-Hydroxypyruvate and benzoate levels were increased and decreased, respectively, suggesting β-hydroxypyruvate production from d-serine. In vitro, β-hydroxypyruvate altered excitatory properties of myenteric neurons and reduced islet insulin content but not GSIS. β-Hydroxypyruvate–to–d-serine ratios were lower in humans with impaired glucose tolerance compared with normal glucose tolerance and T2DM. Earlier human studies unmasked a neural relay that amplifies GIP-mediated insulin secretion in a pattern reciprocal to β-hydroxypyruvate–to–d-serine ratios in all groups. Thus, K cells may maintain long-term function of neurons and β-cells by regulating β-hydroxypyruvate levels. PMID:25368100

  3. 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

  4. 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

  5. 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.

  6. 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

  7. Aggressive behavior in humans: Genes and pathways identified through association studies.

    PubMed

    Fernàndez-Castillo, Noèlia; Cormand, Bru

    2016-07-01

    Aggressive behavior has both genetic and environmental components. Many association studies have been performed to identify genetic factors underlying aggressive behaviors in humans. In this review we summarize the previous work performed in this field, considering both candidate gene (CGAS) and genome-wide association studies (GWAS), excluding those performed in samples where the primary diagnosis is a psychiatric or neurological disorder other than an aggression-related phenotype. Subsequently, we have studied the enrichment of pathways and functions in GWAS data. The results of our searches show that most CGAS have identified associations with genes involved in dopaminergic and serotonergic neurotransmission and in hormone regulation. On the other hand, GWAS have not yet identified genome-wide significant associations, but top nominal findings are related to several signaling pathways, such as axon guidance or estrogen receptor signaling, and also to neurodevelopmental processes and synaptic plasticity. Future studies should use larger samples, homogeneous phenotypes and standardized measurements to identify genes that underlie aggressive behaviors in humans. © 2016 Wiley Periodicals, Inc. PMID:26773414

  8. GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway.

    PubMed

    Ishida, Hidekazu; Saba, Rie; Kokkinopoulos, Ioannis; Hashimoto, Masakazu; Yamaguchi, Osamu; Nowotschin, Sonja; Shiraishi, Manabu; Ruchaya, Prashant; Miller, Duncan; Harmer, Stephen; Poliandri, Ariel; Kogaki, Shigetoyo; Sakata, Yasushi; Dunkel, Leo; Tinker, Andrew; Hadjantonakis, Anna-Katerina; Sawa, Yoshiki; Sasaki, Hiroshi; Ozono, Keiichi; Suzuki, Ken; Yashiro, Kenta

    2016-07-26

    A surface marker that distinctly identifies cardiac progenitors (CPs) is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2), specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure. PMID:27396331

  9. 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

  10. 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

  11. Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.

    PubMed

    DeJesus, Rowena; Moretti, Francesca; McAllister, Gregory; Wang, Zuncai; Bergman, Phil; Liu, Shanming; Frias, Elizabeth; Alford, John; Reece-Hoyes, John S; Lindeman, Alicia; Kelliher, Jennifer; Russ, Carsten; Knehr, Judith; Carbone, Walter; Beibel, Martin; Roma, Guglielmo; Ng, Aylwin; Tallarico, John A; Porter, Jeffery A; Xavier, Ramnik J; Mickanin, Craig; Murphy, Leon O; Hoffman, Gregory R; Nyfeler, Beat

    2016-01-01

    SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein. PMID:27351204

  12. 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

  13. Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

    PubMed Central

    DeJesus, Rowena; Moretti, Francesca; McAllister, Gregory; Wang, Zuncai; Bergman, Phil; Liu, Shanming; Frias, Elizabeth; Alford, John; Reece-Hoyes, John S; Lindeman, Alicia; Kelliher, Jennifer; Russ, Carsten; Knehr, Judith; Carbone, Walter; Beibel, Martin; Roma, Guglielmo; Ng, Aylwin; Tallarico, John A; Porter, Jeffery A; Xavier, Ramnik J; Mickanin, Craig; Murphy, Leon O; Hoffman, Gregory R; Nyfeler, Beat

    2016-01-01

    SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein. DOI: http://dx.doi.org/10.7554/eLife.17290.001 PMID:27351204

  14. Modulation of nitrergic signalling pathway by American ginseng attenuates chronic unpredictable stress-induced cognitive impairment, neuroinflammation, and biochemical alterations.

    PubMed

    Rinwa, Puneet; Kumar, Anil

    2014-02-01

    Prolonged stress causes extensive loss of neurons leading to deficits in cognitive performance. Increasing evidence indicates that accumulation of intercellular messenger, nitric oxide (NO), plays a crucial role in the pathogenesis of memory disorders. American ginseng (AG) is known to show protection in different animal models of neurological diseases; however, its exact mechanism of action is not clearly understood. Therefore, the current study was designed to investigate the interaction of AG against chronic unpredictable stress (CUS)-associated behavioral and biochemical alterations and the probable role of nitrergic pathway in this effect. Male Laca mice were exposed to a series of stressors along with drug/vehicle treatment daily for 28 days. CUS paradigm caused significant impairment in both acquisition and retention memory as measured in Morris water maze and elevated plus maze task. This was coupled with alterations in oxidative stress markers, mitochondrial enzyme complex activities, pro-inflammatory cytokine (TNF-α), and acetylcholinesterase levels in the hippocampus as compared with naïve group. Besides, there was a marked increase in serum corticosterone levels. AG (100, 200 mg/kg; p.o.) treatment significantly improved cognitive impairment; reduced TNF-α, acetylcholinesterase, and corticosterone levels; and attenuated oxidative-nitrergic stress. Furthermore, pre-treatment of L-arginine (100 mg/kg; i.p.), a nitric oxide donor, with subeffective dose of AG (100 mg/kg; p.o.) reversed its protective effects. However, L-NAME (10 mg/kg, i.p.), a non-specific NO synthase inhibitor, potentiated the effects of AG. Our findings suggest that modulation of nitrergic signalling cascade is involved in the protective effects of AG against CUS-induced cognitive dysfunction, oxidative stress, and neuroinflammation. PMID:24132508

  15. From L-dopa to dihydroxyphenylacetaldehyde: a toxic biochemical pathway plays a vital physiological function in insects.

    PubMed

    Vavricka, Christopher; Han, Qian; Huang, Yongping; Erickson, Sara M; Harich, Kim; Christensen, Bruce M; Li, Jianyong

    2011-01-01

    One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects. PMID:21283636

  16. From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in Insects

    PubMed Central

    Vavricka, Christopher; Han, Qian; Huang, Yongping; Erickson, Sara M.; Harich, Kim; Christensen, Bruce M.; Li, Jianyong

    2011-01-01

    One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects. PMID:21283636

  17. 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.

  18. 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; Rodríguez, María A.; Corbera, Joan; Muntoni, Francesco; Feng, Lucy; Rivas, Eloy; Torner, Ferran; Gualandi, Francesca; Gomez-Foix, Anna M.; Ferrer, Anna; Ortez, Carlos; Nascimento, Andrés; 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

  19. Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster.

    PubMed

    Okada, Hirokazu; Ebhardt, H Alexander; Vonesch, Sibylle Chantal; Aebersold, Ruedi; Hafen, Ernst

    2016-01-01

    The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, we perform a proteome-wide association study (PWAS) of a complex quantitative phenotype. We quantify the variation of wing imaginal disc proteomes in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. Our study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations. PMID:27582081

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

    PubMed Central

    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; 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 IκB 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

  1. Identifying Work Setting Profile Factors From the Career Pathway Evaluation Program

    PubMed Central

    Sogol, Elliott M.; Brown, Lawrence M.

    2013-01-01

    Objectives. To describe the work factors associated with 28 different career areas as reported by pharmacists who responded to the American Pharmacists Association (APhA) Career Pathway Evaluation Program for Pharmacy Professionals, 2012 Pharmacist Profile Survey Methods. Data from the 1,119 completed survey instruments from the 2012 Pharmacist Profile Survey were analyzed. Exploratory factor analysis was used to identify the underlying factors that best represented respondents’ work setting profiles. Results. Eleven underlying factors were identified for the respondents’ work setting profiles: patient care, application of clinical knowledge, innovation, stress, research, managerial responsibility, work schedule flexibility, job position flexibility, self-actualization, geographic location, and continuity of coworker relationships. Findings revealed variation for these underlying factors among career categories. Conclusion. Variation among pharmacist career types exists. The profiles constructed in this study describe the characteristics of various career paths and can be helpful for decisions regarding educational, experiential, residency, and certification training in pharmacist careers. PMID:24249856

  2. Comprehensive profiling analysis of actively resorbing osteoclasts identifies critical signaling pathways regulated by bone substrate

    PubMed Central

    Purdue, P. Edward; Crotti, Tania N.; Shen, Zhenxin; Swantek, Jennifer; Li, Jun; Hill, Jonathan; Hanidu, Adedayo; Dimock, Janice; Nabozny, Gerald; Goldring, Steven R.; McHugh, Kevin P.

    2014-01-01

    As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis. PMID:25534583

  3. 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

  4. 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

  5. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human

    PubMed Central

    Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H. T.; Moreira, José C. F.; Suresh, Uthra; Chen, Yidong

    2016-01-01

    Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival. PMID:27100653

  6. 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, NFκB, 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

  7. 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

  8. Soldier, civilian, criminal: identifying pathways to offending of ex-armed forces personnel in prison

    PubMed Central

    Wainwright, Verity; McDonnell, Sharon; Lennox, Charlotte; Shaw, Jenny; Senior, Jane

    2016-01-01

    ABSTRACT Little is known about why some ex-armed forces personnel become involved in the criminal justice system, however, they represent the largest known occupational group in prison. In-depth interviews were employed to explore possible pathways to offending. Twenty ex-armed forces personnel in prison were recruited from five prisons in England. Data were analysed using a combination of thematic analysis and constant comparison methods rooted in grounded theory. Four predominant themes were identified: experiences of trauma and adversity; belonging; impulsivity and creating a soldier. Participants had experienced a number of traumatic incidents and adversity in their lives, encompassing pre, during and post-service but felt a sense of belonging in the armed forces. Participants demonstrated impulsivity in a number of areas with links to both their service in the armed forces and offending behaviour. The creation of the identity of ‘soldier’ was perceived to impact participants’ lives in a number of ways, including their offending, alcohol use and coping with trauma. The interplay of these themes and their potential impact on participants’ pathways to offending are discussed. PMID:27570440

  9. 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 (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ 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

  10. 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; Gómez-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

  11. Biochemical and pharmacological assessment of MAP-kinase signaling along pain pathways in experimental rodent models: a potential tool for the discovery of novel antinociceptive therapeutics.

    PubMed

    Edelmayer, Rebecca M; Brederson, Jill-Desiree; Jarvis, Michael F; Bitner, Robert S

    2014-02-01

    Injury to the peripheral or central nervous system can induce changes within the nervous tissues that promote a state of sensitization that may underlie conditions of pathological chronic pain. A key biochemical event in the initiation and maintenance of peripheral and central neuronal sensitization associated with chronic pain is the phosphorylation and subsequent activation of mitogen-activated protein kinases (MAPKs) and immediate early gene transcription factors, in particular cAMP-response element binding protein (CREB). In this commentary we review the preclinical data that describe anatomical and mechanistic aspects of nociceptive-induced signaling along nociceptive pathways including peripheral cutaneous axons, the dorsal root ganglia, spinal cord dorsal horn and cerebral cortex. In addition to the regional manifestation of nociceptive signaling, investigations have attempted to elucidate the cellular origin of biochemical nociceptive processing in which communication, i.e. cross-talk between neurons and glia is viewed as an essential component of pathogenic pain development. Here, we outline a research strategy by which nociceptive-induced cellular signaling in experimental pain models, specifically MAPK and CREB phosphorylation can be utilized to provide mechanistic insight into drug-target interaction along the nociceptive pathways. We describe a series of studies using nociceptive inflammatory and neuropathic pain models to investigate the effects of known pain therapeutics on nociceptive-induced biochemical signaling and present this as a complementary research strategy for assessing antinociceptive activity useful in the preclinical development of novel pain therapeutics. PMID:24300134

  12. Allelic diversity in MCAD deficiency: the biochemical classification of 54 variants identified during 5 years of ACADM sequencing.

    PubMed

    Smith, Emily H; Thomas, Cheryl; McHugh, David; Gavrilov, Dimitar; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia; Matern, Dietrich; Highsmith, W Edward; Oglesbee, Devin

    2010-07-01

    Medium-chain acyl-coA dehydrogenase (MCAD) deficiency is a commonly detected fatty acid oxidation disorder and its diagnosis relies on both biochemical and molecular analyses. Over a 5-year period, sequencing all 12 exons of the MCAD gene (ACADM) in our laboratory revealed a total of 54 variants in 549 subjects analyzed. As most molecular ACADM testing is referred for the follow-up of an abnormal newborn screening result obtained from an asymptomatic newborn, the identification of a novel DNA variant, or "variant of unknown significance (VUS)," presents clinicians with a dilemma. Frequently, the results of molecular analyses are correlated to biochemical findings, such as the concentration of octanoylcarnitine (C8) in plasma and the excretion of hexanoylglycine (HG) in urine. Here, we describe the classification of genotypes harboring at least one VUS through the comparison of C8 and HG values measured in individuals who are carriers of, or affected with, MCAD deficiency on the basis of the following genotypes: c.985A>G/wildtype, c.199T>C/c.985A>G and c.985A>G/c.985A>G. Our findings emphasize the importance of obtaining both plasma and urine when following up positive newborn screening results and may influence the way physicians counsel their asymptomatic patients about MCAD deficiency after genetic analysis. PMID:20434380

  13. 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

  14. 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

  15. A field test of a biochemical key to identify members of the Anopheles gambiae group of species in north-east Tanzania.

    PubMed

    Marchand, R P; Mnzava, A E

    1985-06-01

    The usefulness of a proposed biochemical key to the species of the Anopheles gambiae complex is tested against the standard chromosomal method in N.E. Tanzania. The chromosomal identifications indicate the presence of three sibling species of the complex in this area, partly in coexisting populations. The electromorph frequencies at the diagnostic enzyme loci, octanol-dehydrogenase (Odh) and superoxide dismutase (Sod), show neither significant geographical nor seasonal variation. It is confirmed that A. merus can be distinguished unambiguously on its Sod genotype. A. gambiae s.s. and A. arabiensis can be identified, using the differences in their Odh polymorphism, with an average error of misidentification of 1 in 300. It is concluded that the biochemical key is sufficiently reliable to be a valuable additional taxonomic tool in the study area. PMID:3906146

  16. 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 1585.39 μM. Under these conditions, approximately 99% of the DMS was degraded within 30 h 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 0.17 h(-1) and 0.63 gs gx(-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

  17. 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

  18. Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

    PubMed

    Blokhuis, Anna M; Koppers, Max; Groen, Ewout J N; van den Heuvel, Dianne M A; Dini Modigliani, Stefano; Anink, Jasper J; Fumoto, Katsumi; van Diggelen, Femke; Snelting, Anne; Sodaar, Peter; Verheijen, Bert M; Demmers, Jeroen A A; Veldink, Jan H; Aronica, Eleonora; Bozzoni, Irene; den Hertog, Jeroen; van den Berg, Leonard H; Pasterkamp, R Jeroen

    2016-08-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations. PMID:27164932

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

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

    PubMed

    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

  5. 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

  6. 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

  7. Identifying biological pathways that underlie primordial short stature using network analysis

    PubMed Central

    Hanson, Dan; Stevens, Adam; Murray, Philip G; Black, Graeme C M; Clayton, Peter E

    2014-01-01

    Mutations in CUL7, OBSL1 and CCDC8, leading to disordered ubiquitination, cause one of the commonest primordial growth disorders, 3-M syndrome. This condition is associated with i) abnormal p53 function, ii) GH and/or IGF1 resistance, which may relate to failure to recycle signalling molecules, and iii) cellular IGF2 deficiency. However the exact molecular mechanisms that may link these abnormalities generating growth restriction remain undefined. In this study, we have used immunoprecipitation/mass spectrometry and transcriptomic studies to generate a 3-M ‘interactome’, to define key cellular pathways and biological functions associated with growth failure seen in 3-M. We identified 189 proteins which interacted with CUL7, OBSL1 and CCDC8, from which a network including 176 of these proteins was generated. To strengthen the association to 3-M syndrome, these proteins were compared with an inferred network generated from the genes that were differentially expressed in 3-M fibroblasts compared with controls. This resulted in a final 3-M network of 131 proteins, with the most significant biological pathway within the network being mRNA splicing/processing. We have shown using an exogenous insulin receptor (INSR) minigene system that alternative splicing of exon 11 is significantly changed in HEK293 cells with altered expression of CUL7, OBSL1 and CCDC8 and in 3-M fibroblasts. The net result is a reduction in the expression of the mitogenic INSR isoform in 3-M syndrome. From these preliminary data, we hypothesise that disordered ubiquitination could result in aberrant mRNA splicing in 3-M; however, further investigation is required to determine whether this contributes to growth failure. PMID:24711643

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

    PubMed

    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

    2015-10-20

    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-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

  9. 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 HK–RR 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 α1 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

  10. Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: genetic and biochemical characterization of two crustacean cyclooxygenases.

    PubMed

    Varvas, Külliki; Kurg, Reet; Hansen, Kristella; Järving, Reet; Järving, Ivar; Valmsen, Karin; Lõhelaid, Helike; Samel, Nigulas

    2009-12-01

    Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world. PMID:19854273

  11. 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

  12. Urine metabolomic analysis identifies potential biomarkers and pathogenic pathways in kidney cancer.

    PubMed

    Kim, Kyoungmi; Taylor, Sandra L; Ganti, Sheila; Guo, Lining; Osier, Michael V; Weiss, Robert H

    2011-05-01

    Kidney cancer is the seventh most common cancer in the Western world, its incidence is increasing, and it is frequently metastatic at presentation, at which stage patient survival statistics are grim. In addition, there are no useful biofluid markers for this disease, such that diagnosis is dependent on imaging techniques that are not generally used for screening. In the present study, we use metabolomics techniques to identify metabolites in kidney cancer patients' urine, which appear at different levels (when normalized to account for urine volume and concentration) from the same metabolites in nonkidney cancer patients. We found that quinolinate, 4-hydroxybenzoate, and gentisate are differentially expressed at a false discovery rate of 0.26, and these metabolites are involved in common pathways of specific amino acid and energetic metabolism, consistent with high tumor protein breakdown and utilization, and the Warburg effect. When added to four different (three kidney cancer-derived and one "normal") cell lines, several of the significantly altered metabolites, quinolinate, α-ketoglutarate, and gentisate, showed increased or unchanged cell proliferation that was cell line-dependent. Further evaluation of the global metabolomics analysis, as well as confirmation of the specific potential biomarkers using a larger sample size, will lead to new avenues of kidney cancer diagnosis and therapy. PMID:21348635

  13. 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

  14. 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.

  15. 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

  16. Joint GWAS Analysis: Comparing similar GWAS at different genomic resolutions identifies novel pathway associations with six complex diseases

    PubMed Central

    McGeachie, Michael J.; Clemmer, George L.; Lasky-Su, Jessica; Dahlin, Amber; Raby, Benjamin A.; Weiss, Scott T.

    2014-01-01

    We show here that combining two existing genome wide association studies (GWAS) yields additional biologically relevant information, beyond that obtained by either GWAS separately. We propose Joint GWAS Analysis, a method that compares a pair of GWAS for similarity among the top SNP associations, top genes identified, gene functional clusters, and top biological pathways. We show that Joint GWAS Analysis identifies additional enriched biological pathways that would be missed by traditional Single-GWAS analysis. Furthermore, we examine the similarities of six complex genetic disorders at the SNP-level, gene-level, gene-cluster-level, and pathway-level. We make concrete hypotheses regarding novel pathway associations for several complex disorders considered, based on the results of Joint GWAS Analysis. Together, these results demonstrate that common complex disorders share substantially more genomic architecture than has been previously realized and that the meta-analysis of GWAS needs not be limited to GWAS of the same phenotype to be informative. PMID:25838990

  17. A Powerful Procedure for Pathway-Based Meta-analysis Using Summary Statistics Identifies 43 Pathways Associated with Type II Diabetes in European Populations

    PubMed Central

    Zhang, Han; Wheeler, William; Hyland, Paula L.; Yang, Yifan; Shi, Jianxin; Chatterjee, Nilanjan; Yu, Kai

    2016-01-01

    Meta-analysis of multiple genome-wide association studies (GWAS) has become an effective approach for detecting single nucleotide polymorphism (SNP) associations with complex traits. However, it is difficult to integrate the readily accessible SNP-level summary statistics from a meta-analysis into more powerful multi-marker testing procedures, which generally require individual-level genetic data. We developed a general procedure called Summary based Adaptive Rank Truncated Product (sARTP) for conducting gene and pathway meta-analysis that uses only SNP-level summary statistics in combination with genotype correlation estimated from a panel of individual-level genetic data. We demonstrated the validity and power advantage of sARTP through empirical and simulated data. We conducted a comprehensive pathway-based meta-analysis with sARTP on type 2 diabetes (T2D) by integrating SNP-level summary statistics from two large studies consisting of 19,809 T2D cases and 111,181 controls with European ancestry. Among 4,713 candidate pathways from which genes in neighborhoods of 170 GWAS established T2D loci were excluded, we detected 43 T2D globally significant pathways (with Bonferroni corrected p-values < 0.05), which included the insulin signaling pathway and T2D pathway defined by KEGG, as well as the pathways defined according to specific gene expression patterns on pancreatic adenocarcinoma, hepatocellular carcinoma, and bladder carcinoma. Using summary data from 8 eastern Asian T2D GWAS with 6,952 cases and 11,865 controls, we showed 7 out of the 43 pathways identified in European populations remained to be significant in eastern Asians at the false discovery rate of 0.1. We created an R package and a web-based tool for sARTP with the capability to analyze pathways with thousands of genes and tens of thousands of SNPs. PMID:27362418

  18. A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signalling Pathways during Early Xenopus Embryogenesis

    PubMed Central

    Zhang, Siwei; Li, Jingjing; Lea, Robert; Amaya, Enrique; Dorey, Karel

    2013-01-01

    Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions. PMID:24244509

  19. An integrative data mining approach to identifying adverse outcome pathway signatures.

    PubMed

    Oki, Noffisat O; Edwards, Stephen W

    2016-03-28

    The Adverse Outcome Pathway (AOP) framework is a tool for making biological connections and summarizing key information across different levels of biological organization to connect biological perturbations at the molecular level to adverse outcomes for an individual or population. Computational approaches to explore and determine these connections can accelerate the assembly of AOPs. By leveraging the wealth of publicly available data covering chemical effects on biological systems, computationally-predicted AOPs (cpAOPs) were assembled via data mining of high-throughput screening (HTS) in vitro data, in vivo data and other disease phenotype information. Frequent Itemset Mining (FIM) was used to find associations between the gene targets of ToxCast HTS assays and disease data from Comparative Toxicogenomics Database (CTD) by using the chemicals as the common aggregators between datasets. The method was also used to map gene expression data to disease data from CTD. A cpAOP network was defined by considering genes and diseases as nodes and FIM associations as edges. This network contained 18,283 gene to disease associations for the ToxCast data and 110,253 for CTD gene expression. Two case studies show the value of the cpAOP network by extracting subnetworks focused either on fatty liver disease or the Aryl Hydrocarbon Receptor (AHR). The subnetwork surrounding fatty liver disease included many genes known to play a role in this disease. When querying the cpAOP network with the AHR gene, an interesting subnetwork including glaucoma was identified. While substantial literature exists to support the potential for AHR ligands to elicit glaucoma, it was not explicitly captured in the public annotation information in CTD. The subnetwork from this analysis suggests a cpAOP that includes changes in CYP1B1 expression, which has been previously established in the literature as a primary cause of glaucoma. These case studies highlight the value in integrating multiple data

  20. 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

  1. GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway.

    PubMed

    Bergeron, Francis; Nadeau, Gabriel; Viger, Robert S

    2015-03-01

    GATA4 is an essential transcription factor required for the initiation of genital ridge formation, for normal testicular and ovarian differentiation at the time of sex determination, and for male and female fertility in adulthood. In spite of its crucial roles, the genes and/or gene networks that are ultimately regulated by GATA4 in gonadal tissues remain to be fully understood. This is particularly true for the steroidogenic lineages such as Leydig cells of the testis where many in vitro (promoter) studies have provided good circumstantial evidence that GATA4 is a key regulator of Leydig cell gene expression and steroidogenesis, but formal proof is still lacking. We therefore performed a microarray screening analysis of MA-10 Leydig cells in which Gata4 expression was knocked down using an siRNA strategy. Analysis identified several GATA4-regulated pathways including cholesterol synthesis, cholesterol transport, and especially steroidogenesis. A decrease in GATA4 protein was associated with decreased expression of steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and Star. Gata4 knockdown also led to an important decrease in other novel steroidogenic targets including Srd5a1, Gsta3, Hsd3b1, and Hsd3b6, as well as genes known to participate in cholesterol metabolism such as Scarb1, Ldlr, Soat1, Scap, and Cyp51. Consistent with the decreased expression of these genes, a reduction in GATA4 protein compromised the ability of MA-10 cells to produce steroids both basally and under hormone stimulation. These data therefore provide strong evidence that GATA4 is an essential transcription factor that sits atop of the Leydig cell steroidogenic program. PMID:25504870

  2. 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

  3. Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.

    PubMed

    Drozdov, Ignat; Svejda, Bernhard; Gustafsson, Bjorn I; Mane, Shrikant; Pfragner, Roswitha; Kidd, Mark; Modlin, Irvin M

    2011-01-01

    Small intestinal (SI) neuroendocrine tumors (NET) are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including 'Nervous system development', 'Immune response', and 'Cell-cycle'. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE) transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations). All were up-regulated (p<0.035) with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10(-5) M) significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10(-5) M) stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D(2) and Serotonin [5-HT(2)] receptor agonist, 10(-6) M) stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8-2-fold for isoproterenol and forskolin). Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional effects are

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

    PubMed Central

    Avsar, Timucin; Durası, İlknur Melis; Uygunoğlu, Uğur; Tütüncü, Melih; Demirci, Nuri Onat; Saip, Sabahattin; Sezerman, O. Uğur; Siva, Aksel; Tahir Turanlı, 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

  5. Identifiable Achatina giant neurones: their localizations in ganglia, axonal pathways and pharmacological features.

    PubMed

    Takeuchi, H; Araki, Y; Emaduddin, M; Zhang, W; Han, X Y; Salunga, T L; Wong, S M

    1996-01-01

    1. An African giant snail (Achatina fulica Férussac), originally from East Africa, is now found abundantly in tropical and subtropical regions of Asia, including Okinawa in Japan. This is one of the largest land snail species in the world. The Achatina central nervous system is composed of the buccal, cerebral and suboesophageal ganglia. The 37 giant neurones were identified in these ganglia by the series of studies conducted over about 20 years. The identifications were made by the localization of these neurones in the ganglia, their axonal pathways and their pharmacological features. 2. In the left buccal ganglion, the four giant neurones, d-LBAN, d-LBMB, d-LBCN and d-LBPN, were identified. In the left and right cerebral ganglia, d-LCDN, d-RCDN, v-LCDN and v-RCDN were identified. The suboesophageal ganglia are further composed of the left and right parietal, the visceral, the left and right pleural, and the left and right pedal ganglia. In the right parietal ganglion, PON, TAN, TAN-2, TAN-3, RAPN, d-RPLN, BAPN, LPPN, LBPN, LAPN and v-RPLN were identified. In the visceral ganglion, VIN, FAN, INN, d-VLN, v-VLN, v-VAN, LVMN, RVMN and v-VNAN were identified. In the left parietal ganglion, v-LPSN was identified. In the left and right pedal ganglia, LPeNLN, RPeNLN, d-LPeLN, d-LPeCN, d-RPeAN, d-LPeDN, d-LPeMN and d-LPeEN were identified. 3. Of the small molecule compounds tested, dopamine, 5-hydroxytryptamine, GABA, L-glutamic acid, threo- or erythro-beta-hydroxy-L-glutamic acid were effective on the Achatina giant neurones. We suppose that these compounds act as the neurotransmitters for these neurones. 4. Of the neuroactive peptides, achatin-I(Gly-D-Phe-Ala-Asp). APGW-amide(Ala-Pro-Gly-Trp-NH2) and Achatina cardioexcitatory peptide (ACEP-1)(Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2) were proposed as neurotransmitters, because these were effective on the Achatina giant neurones and their presence was demonstrated in the Achatina ganglia. Further, myomodulin (Pro

  6. Integrated Pathway-Based Approach Identifies Association between Genomic Regions at CTCF and CACNB2 and Schizophrenia

    PubMed Central

    Zapatka, Marc; Frank, Josef; Witt, Stephanie H.; Mühleisen, Thomas W.; Treutlein, Jens; Strohmaier, Jana; Meier, Sandra; Degenhardt, Franziska; Giegling, Ina; Ripke, Stephan; Leber, Markus; Lange, Christoph; Schulze, Thomas G.; Mössner, Rainald; Nenadic, Igor; Sauer, Heinrich; Rujescu, Dan; Maier, Wolfgang; Børglum, Anders; Ophoff, Roel; Cichon, Sven; Nöthen, Markus M.; Rietschel, Marcella; Mattheisen, Manuel; Brors, Benedikt

    2014-01-01

    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

  7. 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; Mühleisen, Thomas W; Treutlein, Jens; Strohmaier, Jana; Meier, Sandra; Degenhardt, Franziska; Giegling, Ina; Ripke, Stephan; Leber, Markus; Lange, Christoph; Schulze, Thomas G; Mössner, Rainald; Nenadic, Igor; Sauer, Heinrich; Rujescu, Dan; Maier, Wolfgang; Børglum, Anders; Ophoff, Roel; Cichon, Sven; Nöthen, 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

  8. 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...

  9. 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

  10. 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-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

  11. 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.

  12. 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…

  13. Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N'-diacetylbacillosamine.

    PubMed

    Hartley, Meredith D; Morrison, Michael J; Aas, Finn Erik; Børud, Bente; Koomey, Michael; Imperiali, Barbara

    2011-06-01

    The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets. PMID:21542610

  14. Identifying pathways and processes affecting nitrate and orthophosphate inputs to streams in agricultural watersheds

    USGS Publications Warehouse

    Tesoriero, A.J.; Duff, J.H.; Wolock, D.M.; Spahr, N.E.; Almendinger, J.E.

    2009-01-01

    Understanding nutrient pathways to streams will improve nutrient management strategies and estimates of the time lag between when changes in land use practices occur and when water quality effects that result from these changes are observed. Nitrate and orthophosphate (OP) concentrations in several environmental compartments were examined in watersheds having a range of base flow index (BFI) values across the continental United States to determine the dominant pathways for water and nutrient inputs to streams. Estimates of the proportion of stream nitrate that was derived from groundwater increased as BFI increased. Nitrate concentration gradients between groundwater and surface water further supported the groundwater source of nitrate in these high BFI streams. However, nitrate concentrations in stream-bed pore water in all settings were typically lower than stream or upland groundwater concentrations, suggesting that nitrate discharge to streams was not uniform through the bed. Rather, preferential pathways (e.g., springs, seeps) may allow high nitrate groundwater to bypass sites of high biogeochemical transformation. Rapid pathway compartments (e.g., overland flow, tile drains) had OP concentrations that were typically higher than in streams and were important OP conveyers in most of these watersheds. In contrast to nitrate, the proportion of stream OP that is derived from ground water did not systematically increase as BFI increased. While typically not the dominant source of OP, groundwater discharge was an important pathway of OP transport to streams when BFI values were very high and when geochemical conditions favored OP mobility in groundwater. Copyright ?? 2009 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.

  15. 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 1.58Å 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 × 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

  16. 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

  17. 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

    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

  18. 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

  19. Preventing the development of SLE: identifying risk factors and proposing pathways for clinical care.

    PubMed

    Choi, M Y; Barber, M R W; Barber, C E H; Clarke, A E; Fritzler, M J

    2016-07-01

    Although challenging, developing evidence-based approaches to an early and accurate diagnosis of systemic lupus erythematosus is a key approach to preventing disease and lupus-associated morbidity and mortality. Advances in our understanding of preclinical and incomplete lupus erythematosus have enabled the identification of risk factors that may predict disease and the development of potential strategies aimed at primary prevention. Emerging data support the notion that there is a temporal disease progression from initial asymptomatic autoimmunity (preclinical lupus) through early clinical features of the disease (incomplete lupus erythematosus) to finally becoming fully classifiable systemic lupus erythematosus (complete lupus erythematosus). Here, we review the demographic, clinical, biomarker as well as genetic and environmental features that are reported to increase the risk of disease progression. Based on these risk factors, we propose a clinical care pathway for patients with early disease. We envisage that such a pathway, through early identification of disease, may improve patient outcomes, while reducing health care costs. PMID:27252260

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

    PubMed

    Pérez, Alexander J; Wesche, Frank; Adihou, Hélène; 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

  1. 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

  2. siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

    PubMed Central

    Bett, John S.; Ibrahim, Adel F. M.; Garg, Amit K.; Rocha, Sonia; Hay, Ronald T.

    2014-01-01

    Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.  To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA “ubiquitome” library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.  Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.  An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening.  The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.  Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments. PMID:24893647

  3. 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

  4. 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

  5. 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

  6. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53.

    PubMed

    Datta, Arindam; Dey, Sanjib; Das, Pijush; Alam, Sk Kayum; Roychoudhury, Susanta

    2016-06-01

    Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF) properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC) harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR) treated SW480 cells expressing mutant p53(R273H) (GEO#: GSE77533). We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53. PMID:27114909

  7. Kinetic properties of a MNB/DYRK1A mutant suitable for the elucidation of biochemical pathways.

    PubMed

    Adayev, Tatyana; Chen-Hwang, Mo-Chou; Murakami, Noriko; Wegiel, Jerzy; Hwang, Yu-Wen

    2006-10-01

    Minibrain kinase/dual-specificity tyrosine phosphorylation regulated kinase 1A (MNB/DYRK1A) is a proline/arginine-directed serine/threonine kinase implicated in the learning deficits of Down syndrome. Epigallocatechin-3-gallate (EGCG), the major tea polyphenolic compound, is a potent MNB/DYRK1A inhibitor. In this study, we investigated the mechanism of EGCG inhibition of MNB/DYRK1A using a combination of genetic and biochemical approaches. In the testing system using MNB/DYRK1A-promoted Gli 1-dependent transcription as the readout, NIH3T3 cells expressing EGCG resistant MNB/DYRK1A mutant R21 were found to acquire EGCG resistance for a wide range of drug concentrations. Mutant R21 harbors a single K465R substitution, which produces a 3-fold gain in the EGCG resistance in vitro. However, the gain in the EGCG resistance alone cannot fully interpret the effectiveness of mutant R21 in suppressing EGCG in cultured cells. Kinetic analysis suggests that EGCG functions as a noncompetitive inhibitor against ATP. Interestingly, the K465R mutation changes the mode of EGCG inhibition on MNB/DYRK1A so that it becomes a competitive inhibitor against ATP. This competitive mode of EGCG inhibition coupled with high intracellular ATP concentrations and an elevated EGCG resistance are likely to be the basis for the resistant property of mutant R21 in cultured cells. The K465R mutation apparently transforms the intramolecular interactions required for MNB/DYRK1A catalysis. This mutant would also be valuable for the elucidation of the mechanisms of MNB/DYRK1A-catalyzed reaction. PMID:17002300

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

    PubMed Central

    Ślesak, 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 peroxide—Oxygen—Origin of life—Photosynthesis—Superoxide dismutase—Superoxide reductase. Astrobiology 12, 775–784. PMID:22970865

  9. Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

    PubMed Central

    Smith, Catherine E.; Mendillo, Marc L.; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S.; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

    2013-01-01

    Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway. PMID:24204293

  10. 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

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

    PubMed Central

    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

  12. 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

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

    PubMed Central

    RUAN, XIYUN; LI, HONGYUN; LIU, BO; CHEN, JIE; ZHANG, SHIBAO; SUN, ZEQIANG; LIU, SHUANGQING; SUN, FAHAI; LIU, QINGYONG

    2015-01-01

    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

  14. 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

  15. Deep sequencing and in silico analyses identify MYB-regulated gene networks and signaling pathways in pancreatic cancer

    PubMed Central

    Azim, Shafquat; Zubair, Haseeb; Srivastava, Sanjeev K.; Bhardwaj, Arun; Zubair, Asif; Ahmad, Aamir; Singh, Seema; Khushman, Moh’d.; Singh, Ajay P.

    2016-01-01

    We have recently demonstrated that the transcription factor MYB can modulate several cancer-associated phenotypes in pancreatic cancer. In order to understand the molecular basis of these MYB-associated changes, we conducted deep-sequencing of transcriptome of MYB-overexpressing and -silenced pancreatic cancer cells, followed by in silico pathway analysis. We identified significant modulation of 774 genes upon MYB-silencing (p < 0.05) that were assigned to 25 gene networks by in silico analysis. Further analyses placed genes in our RNA sequencing-generated dataset to several canonical signalling pathways, such as cell-cycle control, DNA-damage and -repair responses, p53 and HIF1α. Importantly, we observed downregulation of the pancreatic adenocarcinoma signaling pathway in MYB-silenced pancreatic cancer cells exhibiting suppression of EGFR and NF-κB. Decreased expression of EGFR and RELA was validated by both qPCR and immunoblotting and they were both shown to be under direct transcriptional control of MYB. These observations were further confirmed in a converse approach wherein MYB was overexpressed ectopically in a MYB-null pancreatic cancer cell line. Our findings thus suggest that MYB potentially regulates growth and genomic stability of pancreatic cancer cells via targeting complex gene networks and signaling pathways. Further in-depth functional studies are warranted to fully understand MYB signaling in pancreatic cancer. PMID:27354262

  16. Deep sequencing and in silico analyses identify MYB-regulated gene networks and signaling pathways in pancreatic cancer.

    PubMed

    Azim, Shafquat; Zubair, Haseeb; Srivastava, Sanjeev K; Bhardwaj, Arun; Zubair, Asif; Ahmad, Aamir; Singh, Seema; Khushman, Moh'd; Singh, Ajay P

    2016-01-01

    We have recently demonstrated that the transcription factor MYB can modulate several cancer-associated phenotypes in pancreatic cancer. In order to understand the molecular basis of these MYB-associated changes, we conducted deep-sequencing of transcriptome of MYB-overexpressing and -silenced pancreatic cancer cells, followed by in silico pathway analysis. We identified significant modulation of 774 genes upon MYB-silencing (p < 0.05) that were assigned to 25 gene networks by in silico analysis. Further analyses placed genes in our RNA sequencing-generated dataset to several canonical signalling pathways, such as cell-cycle control, DNA-damage and -repair responses, p53 and HIF1α. Importantly, we observed downregulation of the pancreatic adenocarcinoma signaling pathway in MYB-silenced pancreatic cancer cells exhibiting suppression of EGFR and NF-κB. Decreased expression of EGFR and RELA was validated by both qPCR and immunoblotting and they were both shown to be under direct transcriptional control of MYB. These observations were further confirmed in a converse approach wherein MYB was overexpressed ectopically in a MYB-null pancreatic cancer cell line. Our findings thus suggest that MYB potentially regulates growth and genomic stability of pancreatic cancer cells via targeting complex gene networks and signaling pathways. Further in-depth functional studies are warranted to fully understand MYB signaling in pancreatic cancer. PMID:27354262

  17. 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

  18. Integrative Analysis of Metabolomics and Transcriptomics Data: A Unified Model Framework to Identify Underlying System Pathways

    PubMed Central

    Brink-Jensen, Kasper; Bak, Søren; Jørgensen, Kirsten; Ekstrøm, Claus Thorn

    2013-01-01

    The abundance of high-dimensional measurements in the form of gene expression and mass spectroscopy calls for models to elucidate the underlying biological system. For widely studied organisms like yeast, it is possible to incorporate prior knowledge from a variety of databases, an approach used in several recent studies. However if such information is not available for a particular organism these methods fall short. In this paper we propose a statistical method that is applicable to a dataset consisting of Liquid Chromatography-Mass Spectroscopy (LC-MS) and gene expression (DNA microarray) measurements from the same samples, to identify genes controlling the production of metabolites. Due to the high dimensionality of both LC-MS and DNA microarray data, dimension reduction and variable selection are key elements of the analysis. Our proposed approach starts by identifying the basis functions (“building blocks”) that constitute the output from a mass spectrometry experiment. Subsequently, the weights of these basis functions are related to the observations from the corresponding gene expression data in order to identify which genes are associated with specific patterns seen in the metabolite data. The modeling framework is extremely flexible as well as computationally fast and can accommodate treatment effects and other variables related to the experimental design. We demonstrate that within the proposed framework, genes regulating the production of specific metabolites can be identified correctly unless the variation in the noise is more than twice that of the signal. PMID:24086255

  19. 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

  20. 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

  1. A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription.

    PubMed Central

    Grépin, C; Dagnino, L; Robitaille, L; Haberstroh, L; Antakly, T; Nemer, M

    1994-01-01

    In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis. Images PMID:8164667

  2. 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

  3. Systems approaches to unraveling plant metabolism: identifying biosynthetic genes of secondary metabolic pathways.

    PubMed

    Spiering, Martin J; Kaur, Bhavneet; Parsons, James F; Eisenstein, Edward

    2014-01-01

    The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants. PMID:24218220

  4. 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

  5. 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

  6. 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, Tõnu; 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; d’Adamo, 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, Zoltán; 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; Paré, Guillaume; Parker, Alex N; Pedersen, Nancy L; Peeters, Petra HM; Pistis, Giorgio; Plump, Andrew S; Polasek, Ozren; Pop, Victor JM; Psaty, Bruce M; Räikkönen, 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, Gérard; 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, André G; van Duijn, Cornelia M; Völzke, 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, NFκB signalling and mitochondrial dysfunction as biological processes related to timing of menopause. PMID:22267201

  7. 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, Tõnu; 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, Zoltán; 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; Paré, 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; Räikkönen, 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, Gérard; 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, André G; van Duijn, Cornelia M; Völzke, 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-κB signaling and mitochondrial dysfunction as biological processes related to timing of menopause. PMID:22267201

  8. How metal films de-wet substrates : identifying the kinetic pathways and energetic driving forces.

    SciTech Connect

    Thurmer, Konrad; Schmid, Andreas; Sato, Yu; Hamilton, John C.; Saa, Angela; McCarty, Kevin F.; de la Figuera, Juan; Bartelt, Norman Charles

    2008-10-01

    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.

  9. 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 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 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

  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. 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 9p13.3–23.3 (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

  13. Purification of the Prep1 interactome identifies novel pathways regulated by Prep1.

    PubMed

    Díaz, Víctor M; Bachi, Angela; Blasi, Francesco

    2007-08-01

    Prep1 homeodomain transcription factor interacts with Pbx proteins to regulate oculogenesis, angiogenesis, and hematopoiesis in mice. To isolate new Prep1 interactors competing or copurifying with Pbx, we identified proteins copurified with Prep1-TAP by tandem affinity purification (TAP). Prep1-TAP was fully functional and allowed the isolation of a Prep1 proteome from cytoplasm and nucleus, but most interactors were nuclear. The Prep1-TAP complex included Pbx1b, Pbx2, and other nonhomeodomain proteins: p160 Myb-binding protein (p160), beta-actin, NMMHCIIA. PMID:17623278

  14. 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; Mršić, 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 β-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

  15. 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.

  16. Uncovering multiple pathways to substance use: a comparison of methods for identifying population subgroups.

    PubMed

    Dierker, Lisa; Rose, Jennifer; Tan, Xianming; Li, Runze

    2010-12-01

    This paper describes and compares a selection of available modeling techniques for identifying homogeneous population subgroups in the interest of informing targeted substance use intervention. We present a nontechnical review of the common and unique features of three methods: (a) trajectory analysis, (b) functional hierarchical linear modeling (FHLM), and (c) decision tree methods. Differences among the techniques are described, including required data features, strengths and limitations in terms of the flexibility with which outcomes and predictors can be modeled, and the potential of each technique for helping to inform the selection of targets and timing of substance intervention programs. PMID:20844962

  17. Finding novel distinctions between the sAPPα-mediated anabolic biochemical pathways in Autism Spectrum Disorder and Fragile X Syndrome plasma and brain tissue

    PubMed Central

    Ray, Balmiki; Sokol, Deborah K.; Maloney, Bryan; Lahiri, Debomoy K.

    2016-01-01

    Autism spectrum disorder (ASD) and Fragile X syndrome (FXS) are developmental disorders. No validated blood-based biomarkers exist for either, which impedes bench-to-bedside approaches. Amyloid-β (Aβ) precursor protein (APP) and metabolites are usually associated with Alzheimer’s disease (AD). APP cleavage by α-secretase produces potentially neurotrophic secreted APPα (sAPPα) and the P3 peptide fragment. β-site APP cleaving enzyme (BACE1) cleavage produces secreted APPβ (sAPPβ) and intact Aβ. Excess Aβ is potentially neurotoxic and can lead to atrophy of brain regions such as amygdala in AD. By contrast, amygdala is enlarged in ASD but not FXS. We previously reported elevated levels of sAPPα in ASD and FXS vs. controls. We now report elevated plasma Aβ and total APP levels in FXS compared to both ASD and typically developing controls, and elevated levels of sAPPα in ASD and FXS vs. controls. By contrast, plasma and brain sAPPβ and Aβ were lower in ASD vs. controls but elevated in FXS plasma vs. controls. We also detected age-dependent increase in an α-secretase in ASD brains. We report a novel mechanistic difference in APP pathways between ASD (processing) and FXS (expression) leading to distinct APP metabolite profiles in these two disorders. These novel, distinctive biochemical differences between ASD and FXS pave the way for blood-based biomarkers for ASD and FXS. PMID:27212113

  18. Abiogenic hydrocarbon isotopic signatures in granitic rocks: Identifying pathways of formation

    NASA Astrophysics Data System (ADS)

    Potter, Joanna; Salvi, Stefano; Longstaffe, Fred J.

    2013-12-01

    The stability and isotopic composition of hydrocarbons formed in the mantle are controversial subjects. Knowing the range in isotopic compositions of abiogenically-derived hydrocarbons is important for recognising biological signatures in ancient and extra-terrestrial materials. In an effort to enhance this database, stable isotope results are reported here for hydrocarbon-bearing fluid inclusions hosted in two peralkaline igneous complexes from Lovozero, Russia and Strange Lake, Canada. Based on the distribution of isotopic compositions, we propose three pathways for abiogenically-generated hydrocarbons. Type-1 (δ13CCH4 > δ13CCO2 and δ13CC2 +) represents mantle-derived hydrocarbons generated in equilibrium with hyperagpaitic magmas in the upper mantle, and suggests that mantle CH4 and C2H6δ13C and δ2H could have values of ~ - 5.3 and ~ - 112‰, and ~ - 10.8 and - 162‰, respectively. Type-2 (δ13CCO2 > δ13CCH4 > δ13CC2 +) represents Fischer-Tropsch-type hydrocarbon generation in quartz-bearing peralkaline rocks with CO2 as the initial magmatic gas phase. For Type-2, δ13CCO2 values are ~ - 2‰, whereas δ13CCH4 and δ2HCH4 values range from - 32 to - 20‰ and - 170 and - 160‰, respectively. The δ13CC2H6 values are slightly lower than associated δ13CCH4 values. Type-3 (δ13CCO2 > δ13CCH4 < δ13CC2 +) represents an overprint of Type-2, formed during low temperature alteration of quartz-bearing peralkaline rocks. Here, δ13CCO2 values are variable, - 14.0 to - 9.5‰, δ13CCH4 values range from - 30.8 to - 20.8‰ while δ13CC2H6 values are higher than associated δ13CCH4 values. Both Type-2 and Type-3 isotopic compositions mimic patterns normally considered to be thermogenic in origin, thus demonstrating that isotopic data alone cannot be used reliably to distinguish between hydrocarbons of abiogenic versus biogenic origin.

  19. MRI anatomy of parametrial extension to better identify local pathways of disease spread in cervical cancer

    PubMed Central

    Valentini, Anna Lia; Gui, Benedetta; Miccò, Maura; Giuliani, Michela; Rodolfino, Elena; Ninivaggi, Valeria; Iacobucci, Marta; Marino, Marzia; Gambacorta, Maria Antonietta; Testa, Antonia Carla; Zannoni, Gian Franco; Bonomo, Lorenzo

    2016-01-01

    This paper highlights an updated anatomy of parametrial extension with emphasis on magnetic resonance imaging (MRI) assessment of disease spread in the parametrium in patients with locally advanced cervical cancer. Pelvic landmarks were identified to assess the anterior and posterior extensions of the parametria, besides the lateral extension, as defined in a previous anatomical study. A series of schematic drawings and MRI images are shown to document the anatomical delineation of disease on MRI, which is crucial not only for correct image-based three-dimensional radiotherapy but also for the surgical oncologist, since neoadjuvant chemoradiotherapy followed by radical surgery is emerging in Europe as a valid alternative to standard chemoradiation. PMID:27165471

  20. The Robustness of Pathway Analysis in Identifying Potential Drug Targets in Non-Small Cell Lung Carcinoma

    PubMed Central

    Dalby, Andrew; Bailey, Ian

    2014-01-01

    The identification of genes responsible for causing cancers from gene expression data has had varied success. Often the genes identified depend on the methods used for detecting expression patterns, or on the ways that the data had been normalized and filtered. The use of gene set enrichment analysis is one way to introduce biological information in order to improve the detection of differentially expressed genes and pathways. In this paper we show that the use of network models while still subject to the problems of normalization is a more robust method for detecting pathways that are differentially overrepresented in lung cancer data. Such differences may provide opportunities for novel therapeutics. In addition, we present evidence that non-small cell lung carcinoma is not a series of homogeneous diseases; rather that there is a heterogeny within the genotype which defies phenotype classification. This diversity helps to explain the lack of progress in developing therapies against non-small cell carcinoma and suggests that drug development may consider multiple pathways as treatment targets.

  1. Identifying improvements to complex pathways: evidence synthesis and stakeholder engagement in infant congenital heart disease

    PubMed Central

    Crowe, Sonya; Knowles, Rachel; Wray, Jo; Tregay, Jenifer; Ridout, Deborah A; Utley, Martin; Franklin, Rodney; Bull, Catherine L; Brown, Katherine L

    2016-01-01

    Objectives Many infants die in the year following discharge from hospital after surgical or catheter intervention for congenital heart disease (3–5% of discharged infants). There is considerable variability in the provision of care and support in this period, and some families experience barriers to care. We aimed to identify ways to improve discharge and postdischarge care for this patient group. Design A systematic evidence synthesis aligned with a process of eliciting the perspectives of families and professionals from community, primary, secondary and tertiary care. Setting UK. Results A set of evidence-informed recommendations for improving the discharge and postdischarge care of infants following intervention for congenital heart disease was produced. These address known challenges with current care processes and, recognising current resource constraints, are targeted at patient groups based on the number of patients affected and the level and nature of their risk of adverse 1-year outcome. The recommendations include: structured discharge documentation, discharging certain high-risk patients via their local hospital, enhanced surveillance for patients with certain (high-risk) cardiac diagnoses and an early warning tool for parents and community health professionals. Conclusions Our recommendations set out a comprehensive, system-wide approach for improving discharge and postdischarge services. This approach could be used to address challenges in delivering care for other patient populations that can fall through gaps between sectors and organisations. PMID:27266768

  2. Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis

    PubMed Central

    Polachini, Giovana M.; Sobral, Lays M.; Mercante, Ana M. C.; Paes-Leme, Adriana F.; Xavier, Flávia C. A.; Henrique, Tiago; Guimarães, Douglas M.; Vidotto, Alessandra; Fukuyama, Erica E.; Góis-Filho, José F.; Cury, Patricia M.; Curioni, Otávio A.; Michaluart Jr, Pedro; Silva, Adriana M. A.; Wünsch-Filho, Victor; Nunes, Fabio D.; Leopoldino, Andréia M.; Tajara, Eloiza H.

    2012-01-01

    The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the “more-aggressive” group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the “less-aggressive” group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas. PMID:23227181

  3. 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

  4. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    PubMed

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  5. 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

  6. 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

  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. Novel MEK1 Mutation Identified by Mutational Analysis of Epidermal Growth Factor Receptor Signaling Pathway Genes in Lung Adenocarcinoma

    PubMed Central

    Marks, Jenifer L.; Gong, Yixuan; Chitale, Dhananjay; Golas, Ben; McLellan, Michael D.; Kasai, Yumi; Ding, Li; Mardis, Elaine R.; Wilson, Richard K.; Solit, David; Levine, Ross; Michel, Kathrin; Thomas, Roman K.; Rusch, Valerie W.; Ladanyi, Marc; Pao, William

    2008-01-01

    Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non–small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal–regulated kinase (ERK)-1/2 in human 293T cells and to growth factor–independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma. PMID:18632602

  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. Mutation profiling of adenoid cystic carcinomas from multiple anatomical sites identifies mutations in the RAS pathway, but no KIT mutations

    PubMed Central

    Wetterskog, Daniel; Wilkerson, Paul M; Rodrigues, Daniel N; Lambros, Maryou B; Fritchie, Karen; Andersson, Mattias K; Natrajan, Rachael; Gauthier, Arnaud; Di Palma, Silvana; Shousha, Sami; Gatalica, Zoran; Töpfer, Chantal; Vukovic, Vesna; A’Hern, Roger; Weigelt, Britta; Vincent-Salomon, Anne; Stenman, Göran; Rubin, Brian P; Reis-Filho, Jorge S

    2016-01-01

    Aims The majority of adenoid cystic carcinomas (AdCCs), regardless of anatomical site, harbour the MYB–NFIB fusion gene. The aim of this study was to characterize the repertoire of somatic genetic events affecting known cancer genes in AdCCs. Methods and results DNA was extracted from 13 microdissected breast AdCCs, and subjected to a mutation survey using the Sequenom OncoCarta Panel v1.0. Genes found to be mutated in any of the breast AdCCs and genes related to the same canonical molecular pathways, as well as KIT, a proto-oncogene whose protein product is expressed in AdCCs, were sequenced in an additional 68 AdCCs from various anatomical sites by Sanger sequencing. Using the Sequenom MassARRAY platform and Sanger sequencing, mutations in BRAF and HRAS were identified in three and one cases, respectively (breast, and head and neck). KIT, which has previously been reported to be mutated in AdCCs, was also investigated, but no mutations were identified. Conclusions Our results demonstrate that mutations in genes pertaining to the canonical RAS pathway are found in a minority of AdCCs, and that activating KIT mutations are either absent or remarkably rare in these cancers, and unlikely to constitute a driver and therapeutic target for patients with AdCC. PMID:23398044

  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. 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

  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, Régine; 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. Biochemical characterization and selective inhibition of β-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 β-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 λmax 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 μm) 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 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8. PMID:26257333

  16. What makes the lac-pathway switch: identifying the fluctuations that trigger phenotype switching in gene regulatory systems.

    PubMed

    Bhogale, Prasanna M; Sorg, Robin A; Veening, Jan-Willem; Berg, Johannes

    2014-10-01

    Multistable gene regulatory systems sustain different levels of gene expression under identical external conditions. Such multistability is used to encode phenotypic states in processes including nutrient uptake and persistence in bacteria, fate selection in viral infection, cell-cycle control and development. Stochastic switching between different phenotypes can occur as the result of random fluctuations in molecular copy numbers of mRNA and proteins arising in transcription, translation, transport and binding. However, which component of a pathway triggers such a transition is generally not known. By linking single-cell experiments on the lactose-uptake pathway in E. coli to molecular simulations, we devise a general method to pinpoint the particular fluctuation driving phenotype switching and apply this method to the transition between the uninduced and induced states of the lac-genes. We find that the transition to the induced state is not caused only by the single event of lac-repressor unbinding, but depends crucially on the time period over which the repressor remains unbound from the lac-operon. We confirm this notion in strains with a high expression level of the lac-repressor (leading to shorter periods over which the lac-operon remains unbound), which show a reduced switching rate. Our techniques apply to multistable gene regulatory systems in general and allow to identify the molecular mechanisms behind stochastic transitions in gene regulatory circuits. PMID:25245949

  17. 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

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

    PubMed

    Rudolph, Karen D; Troop-Gordon, Wendy; Lambert, Sharon F; Natsuaki, Misaki N

    2014-11-01

    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. Functional genomics identifies five distinct molecular subtypes with clinical relevance and pathways for growth control in epithelial ovarian cancer

    PubMed Central

    Tan, Tuan Zea; Miow, Qing Hao; Huang, Ruby Yun-Ju; Wong, Meng Kang; Ye, Jieru; Lau, Jieying Amelia; Wu, Meng Chu; Bin Abdul Hadi, Luqman Hakim; Soong, Richie; Choolani, Mahesh; Davidson, Ben; Nesland, Jahn M; Wang, Ling-Zhi; Matsumura, Noriomi; Mandai, Masaki; Konishi, Ikuo; Goh, Boon-Cher; Chang, Jeffrey T; Thiery, Jean Paul; Mori, Seiichi

    2013-01-01

    Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups — Epi-A, Epi-B, Mes, Stem-A and Stem-B — exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients. PMID:23666744

  20. A Sulfite Respiration Pathway from Thermus thermophilus and the Key Role of Newly Identified Cytochrome c550 ▿

    PubMed Central

    Robin, Sylvain; Arese, Marzia; Forte, Elena; Sarti, Paolo; Giuffrè, Alessandro; Soulimane, Tewfik

    2011-01-01

    Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium Thermus thermophilus HB8 encoding both a SOR and a diheme c-type cytochrome. The in vitro analysis clearly showed that the newly identified cytochrome c550 acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome c552 to the terminal ba3- and caa3-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in T. thermophilus: sulfite → SORHB8 → cytochrome c550 → cytochrome c552 → ba3 oxidase/caa3 oxidase → O2. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of T. thermophilus. PMID:21665981

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

    PubMed Central

    Erdmann, Frank; Kügler, Sebastian; Blaesse, Peter; Lange, Maren D.; Skryabin, Boris V.; Pape, Hans-Christian; Jüngling, 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

  2. 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.; O’Malley, 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-2–enriched, 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

  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. 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

  5. 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

  6. Whole blood transcriptomics and urinary metabolomics to define adaptive biochemical pathways of high-intensity exercise in 50-60 year old masters athletes.

    PubMed

    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 50-60 year old males: competitive cyclists (athletes, n = 9; VO2peak 59.1±5.2 ml·kg(-1)·min(-1); peak aerobic power 383±39 W) and untrained, minimally active individuals (controls, n = 8; VO2peak 35.9±9.7 ml·kg(-1)·min(-1); peak aerobic power 230±57 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

  7. 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 50–60 year old males: competitive cyclists (athletes, n = 9; VO2peak 59.1±5.2 ml·kg−1·min−1; peak aerobic power 383±39 W) and untrained, minimally active individuals (controls, n = 8; VO2peak 35.9±9.7 ml·kg−1·min−1; peak aerobic power 230±57 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

  8. 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.

  9. Global gene expression profiling of somatic motor neuron populations with different vulnerability identify molecules and pathways of degeneration and protection

    PubMed Central

    Karlsson, Martin; Osborn, Teresia; Ludwig, Wesley

    2010-01-01

    Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration. PMID:20826431

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways

    PubMed Central

    Melo, Mariane B.; Nguyen, Quynh P.; Cordeiro, Cynthia; Hassan, Musa A.; Yang, Ninghan; McKell, Renée; Rosowski, Emily E.; Julien, Lindsay; Butty, Vincent; Dardé, Marie-Laure; Ajzenberg, Daniel; Fitzgerald, Katherine; Young, Lucy H.; Saeij, Jeroen P. J.

    2013-01-01

    Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts. PMID:24367253

  15. Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways.

    PubMed

    Melo, Mariane B; Nguyen, Quynh P; Cordeiro, Cynthia; Hassan, Musa A; Yang, Ninghan; McKell, Renée; Rosowski, Emily E; Julien, Lindsay; Butty, Vincent; Dardé, Marie-Laure; Ajzenberg, Daniel; Fitzgerald, Katherine; Young, Lucy H; Saeij, Jeroen P J

    2013-01-01

    Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts. PMID:24367253

  16. A Kinetic-Model-Based Approach to Identify Malfunctioning Components in Signal Transduction Pathways from Artificial Clinical Data.

    PubMed

    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-α/NF-κB 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-α/NF-κB model was used to obtain the data in which abnormal oscillation dynamic was shown in the profile of NF-κB. 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

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

    PubMed Central

    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 β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

  18. Mutations in TLR/MYD88 pathway identify a subset of young chronic lymphocytic leukemia patients with favorable outcome.

    PubMed

    Martínez-Trillos, Alejandra; Pinyol, Magda; Navarro, Alba; Aymerich, Marta; Jares, Pedro; Juan, Manel; Rozman, María; Colomer, Dolors; Delgado, Julio; Giné, Eva; González-Díaz, Marcos; Hernández-Rivas, Jesús M; Colado, Enrique; Rayón, Consolación; Payer, Angel R; Terol, Maria José; Navarro, Blanca; Quesada, Victor; Puente, Xosé S; Rozman, Ciril; López-Otín, Carlos; Campo, Elías; López-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 κB 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

  19. First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-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 β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. PMID:26218223

  20. Identifying early pathways of risk and resilience: The codevelopment of internalizing and externalizing symptoms and the role of harsh parenting.

    PubMed

    Wiggins, Jillian Lee; Mitchell, Colter; Hyde, Luke W; Monk, Christopher S

    2015-11-01

    Psychological disorders co-occur often in children, but little has been done to document the types of conjoint pathways internalizing and externalizing symptoms may take from the crucial early period of toddlerhood or how harsh parenting may overlap with early symptom codevelopment. To examine symptom codevelopment trajectories, we identified latent classes of individuals based on internalizing and externalizing symptoms across ages 3-9 and found three symptom codevelopment classes: normative symptoms (low), severe-decreasing symptoms (initially high but rapidly declining), and severe symptoms (high) trajectories. Next, joint models examined how parenting trajectories overlapped with internalizing and externalizing symptom trajectories. These trajectory classes demonstrated that, normatively, harsh parenting increased after toddlerhood, but the severe symptoms class was characterized by a higher level and a steeper increase in harsh parenting and the severe-decreasing class by high, stable harsh parenting. In addition, a transactional model examined the bidirectional relationships among internalizing and externalizing symptoms and harsh parenting because they may cascade over time in this early period. Harsh parenting uniquely contributed to externalizing symptoms, controlling for internalizing symptoms, but not vice versa. In addition, internalizing symptoms appeared to be a mechanism by which externalizing symptoms increase. Results highlight the importance of accounting for both internalizing and externalizing symptoms from an early age to understand risk for developing psychopathology and the role harsh parenting plays in influencing these trajectories. PMID:26439075

  1. Identifying molecular genetic features and oncogenic pathways of clear cell renal cell carcinoma through the anatomical (PADUA) scoring system

    PubMed Central

    Lin, Zhiqian; Shi, Guohai; Lin, Xiaozhu; Wu, Zhiyuan; Zhang, Xia; Zhang, Xi

    2016-01-01

    Although the preoperative aspects and dimensions used for the PADUA scoring system were successfully applied in macroscopic clinical practice for renal tumor, the relevant molecular genetic basis remained unclear. To uncover meaningful correlations between the genetic aberrations and radiological features, we enrolled 112 patients with clear cell renal cell carcinoma (ccRCC) whose clinicopathological data, genomics data and CT data were obtained from The Cancer Genome Atlas (TCGA) and The Cancer Imaging Archive (TCIA). Overall PADUA score and several radiological features included in the PADUA system were assigned for each ccRCC. Despite having observed no significant association between the gene mutation frequency and the overall PADUA score, correlations between gene mutations and a few radiological features (tumor rim location and tumor size) were identified. A significant association between rim location and miRNA molecular subtypes was also observed. Survival analysis revealed that tumor size > 7 cm was significantly associated with poor survival. In addition, Gene Set Enrichment Analysis (GSEA) on mRNA expression revealed that the high PADUA score was related to numerous cancer-related networks, especially epithelial to mesenchymal transition (EMT) related pathways. This preliminary analysis of ccRCC revealed meaningful correlations between PADUA anatomical features and molecular basis including genomic aberrations and molecular subtypes. PMID:26848523

  2. 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

  3. 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.

  4. 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.

  5. 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...

  6. 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...

  7. Multiplexing oscillatory biochemical signals.

    PubMed

    de Ronde, Wiet; ten Wolde, Pieter Rein

    2014-04-01

    In recent years it has been increasingly recognized that biochemical signals are not necessarily constant in time and that the temporal dynamics of a signal can be the information carrier. Moreover, it is now well established that the protein signaling network of living cells has a bow-tie structure and that components are often shared between different signaling pathways. Here we show by mathematical modeling that living cells can multiplex a constant and an oscillatory signal: they can transmit these two signals simultaneously through a common signaling pathway, and yet respond to them specifically and reliably. We find that information transmission is reduced not only by noise arising from the intrinsic stochasticity of biochemical reactions, but also by crosstalk between the different channels. Yet, under biologically relevant conditions more than 2 bits of information can be transmitted per channel, even when the two signals are transmitted simultaneously. These observations suggest that oscillatory signals are ideal for multiplexing signals. PMID:24685537

  8. 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

  9. Characterization of a novel β-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of β-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 β-cypermethrin (β-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 β-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of β-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of β-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. β-CY degradation products were analyzed. Results indicated that YAT strain transformed β-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

  10. 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

  11. 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

  12. 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

  13. 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, ...

  14. Proteomic profiling of naïve multiple myeloma patient plasma cells identifies pathways associated with favourable response to bortezomib-based treatment regimens.

    PubMed

    Dytfeld, Dominik; Rosebeck, Shaun; Kandarpa, Malathi; Mayampurath, Anoop; Mellacheruvu, Dattatreya; Alonge, Mattina M; Ngoka, Lambert; Jasielec, Jagoda; Richardson, Paul G; Volchenboum, Samuel; Nesvizhskii, Alexey I; Sreekumar, Arun; Jakubowiak, Andrzej J

    2015-07-01

    Toward our goal of personalized medicine, we comprehensively profiled pre-treatment malignant plasma cells from multiple myeloma patients and prospectively identified pathways predictive of favourable response to bortezomib-based treatment regimens. We utilized two complementary quantitative proteomics platforms to identify differentially-regulated proteins indicative of at least a very good partial response (VGPR) or complete response/near complete response (CR/nCR) to two treatment regimens containing either bortezomib, liposomal doxorubicin and dexamethasone (VDD), or lenalidomide, bortezomib and dexamethasone (RVD). Our results suggest enrichment of 'universal response' pathways that are common to both treatment regimens and are probable predictors of favourable response to bortezomib, including a subset of endoplasmic reticulum stress pathways. The data also implicate pathways unique to each regimen that may predict sensitivity to DNA-damaging agents, such as mitochondrial dysfunction, and immunomodulatory drugs, which was associated with acute phase response signalling. Overall, we identified patterns of tumour characteristics that may predict response to bortezomib-based regimens and their components. These results provide a rationale for further evaluation of the protein profiles identified herein for targeted selection of anti-myeloma therapy to increase the likelihood of improved treatment outcome of patients with newly-diagnosed myeloma. PMID:25824111

  15. Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

    PubMed Central

    Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C.; Fan, Jian-Qiang

    2007-01-01

    Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. PMID:17555407

  16. 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.

    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

  17. A novel biochemical method to identify target genes of individual microRNAs: Identification of a new Caenorhabditis elegans let-7 target

    PubMed Central

    Andachi, Yoshiki

    2008-01-01

    MicroRNAs (miRNAs) are roughly 22-nucleotide regulatory RNAs that play important roles in many developmental and physiological processes. Animal miRNAs down-regulate target genes by forming imperfect base pairs with 3′ untranslated regions (3′ UTRs) of their mRNAs. Thousands of miRNAs have been discovered in several organisms. However, the target genes of almost all of these miRNAs remain to be identified. Here, we describe a method for isolating cDNA clones of target mRNAs that form base pairs in vivo with an endogenous miRNA of interest, in which the cDNAs are synthesized from the mRNAs using the miRNA as a reverse-transcription primer. The application of this method to Caenorhabditis elegans miRNA lin-4 under test conditions yielded many clones of the known target gene lin-14 that correspond to partial sequences 5′ to lin-4 binding sites in the 3′ UTR. The method was also applied to C. elegans miRNA let-7 and a new target gene responsible for the lethal phenotype in let-7 mutants was identified. These results demonstrate that the method is a useful way to identify targets on the basis of base pairing with individual miRNAs. PMID:18824511

  18. A Canonical Correlation Analysis of AIDS Restriction Genes and Metabolic Pathways Identifies Purine Metabolism as a Key Cooperator

    PubMed Central

    Ye, Hanhui; Yuan, Jinjin; Wang, Zhengwu; Huang, Aiqiong; Liu, Xiaolong; Han, Xiao; Chen, Yahong

    2016-01-01

    Human immunodeficiency virus causes a severe disease in humans, referred to as immune deficiency syndrome. Studies on the interaction between host genetic factors and the virus have revealed dozens of genes that impact diverse processes in the AIDS disease. To resolve more genetic factors related to AIDS, a canonical correlation analysis was used to determine the correlation between AIDS restriction and metabolic pathway gene expression. The results show that HIV-1 postentry cellular viral cofactors from AIDS restriction genes are coexpressed in human transcriptome microarray datasets. Further, the purine metabolism pathway comprises novel host factors that are coexpressed with AIDS restriction genes. Using a canonical correlation analysis for expression is a reliable approach to exploring the mechanism underlying AIDS. PMID:27462363

  19. 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.

  20. 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 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

  1. 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

    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

  2. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics.

    PubMed

    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

    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™ 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® 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. PMID:21925528

  3. Use of RNA-seq to identify cardiac genes and gene pathways differentially expressed between dogs with and without dilated cardiomyopathy.

    PubMed

    Friedenberg, Steven G; Chdid, Lhoucine; Keene, Bruce; Sherry, Barbara; Motsinger-Reif, Alison; Meurs, Kathryn M

    2016-07-01

    OBJECTIVE To identify cardiac tissue genes and gene pathways differentially expressed between dogs with and without dilated cardiomyopathy (DCM). ANIMALS 8 dogs with and 5 dogs without DCM. PROCEDURES Following euthanasia, samples of left ventricular myocardium were collected from each dog. Total RNA was extracted from tissue samples, and RNA sequencing was performed on each sample. Samples from dogs with and without DCM were grouped to identify genes that were differentially regulated between the 2 populations. Overrepresentation analysis was performed on upregulated and downregulated gene sets to identify altered molecular pathways in dogs with DCM. RESULTS Genes involved in cellular energy metabolism, especially metabolism of carbohydrates and fats, were significantly downregulated in dogs with DCM. Expression of cardiac structural proteins was also altered in affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that RNA sequencing may provide important insights into the pathogenesis of DCM in dogs and highlight pathways that should be explored to identify causative mutations and develop novel therapeutic interventions. PMID:27347821

  4. Whole Genome Pathway Analysis Identifies an Association of Cadmium Response Gene Loss with Copy Number Variation in Mutant p53 Bearing Uterine Endometrial Carcinomas

    PubMed Central

    Stupack, Dwayne G

    2016-01-01

    Background Massive chromosomal aberrations are a signature of advanced cancer, although the factors promoting the pervasive incidence of these copy number alterations (CNAs) are poorly understood. Gatekeeper mutations, such as p53, contribute to aneuploidy, yet p53 mutant tumors do not always display CNAs. Uterine Corpus Endometrial Carcinoma (UCEC) offers a unique system to begin to evaluate why some cancers acquire high CNAs while others evolve another route to oncogenesis, since about half of p53 mutant UCEC tumors have a relatively flat CNA landscape and half have 20–90% of their genome altered in copy number. Methods We extracted copy number information from 68 UCEC genomes mutant in p53 by the GISTIC2 algorithm. GO term pathway analysis, via GOrilla, was used to identify suppressed pathways. Genes within these pathways were mapped for focal or wide distribution. Deletion hotspots were evaluated for temporal incidence. Results Multiple pathways contributed to the development of pervasive CNAs, including developmental, metabolic, immunological, cell adhesion and cadmium response pathways. Surprisingly, cadmium response pathway genes are predicted as the earliest loss events within these tumors: in particular, the metallothionein genes involved in heavy metal sequestration. Loss of cadmium response genes were associated with copy number changes and poorer prognosis, contrasting with 'copy number flat' tumors which instead exhibited substantive mutation. Conclusion Metallothioneins are lost early in the development of high CNA endometrial cancer, providing a potential mechanism and biological rationale for increased incidence of endometrial cancer with cadmium exposure. Developmental and metabolic pathways are altered later in tumor progression. PMID:27391266

  5. Three-dimensional pharmacophore design and biochemical screening identifies substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction.

    PubMed

    Reddy, Tummala R K; Li, Chan; Fischer, Peter M; Dekker, Lodewijk V

    2012-08-01

    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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. Genome-wide genetic screen identified the link between dG9a and epidermal growth factor receptor signaling pathway in vivo.

    PubMed

    Shimaji, Kouhei; Konishi, Takahiro; Yoshida, Hideki; Kimura, Hiroshi; Yamaguchi, Masamitsu

    2016-08-01

    G9a is one of the histone H3 Lys 9 (H3K9) specific methyltransferases first identified in mammals. Drosophila G9a (dG9a) has been reported to induce H3K9 dimethylation in vivo, and the target genes of dG9a were identified during embryonic and larval stages. Although dG9a is important for a variety of developmental processes, the link between dG9a and signaling pathways are not addressed yet. Here, by genome-wide genetic screen, taking advantage of the rough eye phenotype of flies that over-express dG9a in eye discs, we identified 16 genes that enhanced the rough eye phenotype induced by dG9a over-expression. These 16 genes included Star, anterior open, bereft and F-box and leucine-rich repeat protein 6 which are components of epidermal growth factor receptor (EGFR) signaling pathway. When dG9a over-expression was combined with mutation of Star, differentiation of R7 photoreceptors in eye imaginal discs as well as cone cells and pigment cells in pupal retinae was severely inhibited. Furthermore, the dG9a over-expression reduced the activated ERK signals in eye discs. These data demonstrate a strong genetic link between dG9a and the EGFR signaling pathway. PMID:27343629

  12. 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

  13. 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.

  14. 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.

  15. Pathway alignment: application to the comparative analysis of glycolytic enzymes.

    PubMed Central

    Dandekar, T; Schuster, S; Snel, B; Huynen, M; Bork, P

    1999-01-01

    Comparative analysis of metabolic pathways in different genomes yields important information on their evolution, on pharmacological targets and on biotechnological applications. In this study on glycolysis, three alternative ways of comparing biochemical pathways are combined: (1) analysis and comparison of biochemical data, (2) pathway analysis based on the concept of elementary modes, and (3) a comparative genome analysis of 17 completely sequenced genomes. The analysis reveals a surprising plasticity of the glycolytic pathway. Isoenzymes in different species are identified and compared; deviations from the textbook standard are detailed. Several potential pharmacological targets and by-passes (such as the Entner-Doudoroff pathway) to glycolysis are examined and compared in the different species. Archaean, bacterial and parasite specific adaptations are identified and described. PMID:10493919

  16. Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

    PubMed

    Oudhoff, Menno J; Antignano, Frann; Chenery, Alistair L; Burrows, Kyle; Redpath, Stephen A; Braam, Mitchell J; Perona-Wright, Georgia; Zaph, Colby

    2016-09-01

    The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7-/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection. PMID:27598373

  17. 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 Bhinder–Djaballah 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

  18. 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

  19. 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

  20. Systematic CpG Islands Methylation Profiling of Genes in the Wnt Pathway in Epithelial Ovarian Cancer Identifies Biomarkers of Progression-Free Survival

    PubMed Central

    Dai, Wei; Teodoridis, Jens M.; Zeller, Constanze; Graham, Janet; Hersey, Jenny; Flanagan, James M.; Stronach, Euan; Millan, David W.; Siddiqui, Nadeem; Paul, Jim; Brown, Robert

    2011-01-01

    Purpose Wnt pathways control key biological processes that potentially impact on tumour progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGIs) of Wnt pathway genes in ovarian tumours at presentation and identify biomarkers of patient progression-free survival (PFS). Experimental Design Epithelial ovarian tumours (screening study n=120, validation study n=61) prospectively collected through a cohort study, were analysed by differential methylation hybridisation (DMH) at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and progression free survival was examined by Cox proportional hazards model. Results DNA methylation is associated with PFS at 20/302 loci (p<0.05, n=111), with 5 loci significant at FDR<10%. 11/20 loci retain significance in an independent validation cohort (n=48,p≤0.05,FDR≤10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1 and NKD1 genes, are independent from clinical parameters (adjusted p<0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS (HR=2.09; 95%CI (1.39, 3.15); permutation test p<0.005). Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1 and ROCK1 is an indicator of early disease relapse in an independent ovarian tumour cohort (n=311, adjusted p<0.05). Conclusions The data highlights the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identifies methylation at NKD1 and DVL1 as independent predictors of PFS. PMID:21459799

  1. Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model

    SciTech Connect

    Fukunaga, Satoki; Kakehashi, Anna; Sumida, Kayo; Kushida, Masahiko; Asano, Hiroyuki; Gi, Min; Wanibuchi, Hideki

    2015-08-01

    To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective of initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis. - Highlights: • We analyzed bleomycin-induced pulmonary fibrosis in the rat. • Integrative analyses of miRNA microarray and proteomics were conducted. • We determined the alterations of miRNAs and their potential target proteins. • The alterations may control biological functions and pathways in pulmonary fibrosis. • Our result may provide new insights of pulmonary fibrosis.

  2. 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

  3. 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

    Schneider, Anna; Corona, Angela; Spöring, Imke; Jordan, Mareike; Buchholz, Bernd; Maccioni, Elias; Di Santo, Roberto; Bodem, Jochen; Tramontano, Enzo; Wöhrl, Birgitta M

    2016-03-18

    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

  4. Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration.

    PubMed

    Koenekoop, Robert K; Wang, Hui; Majewski, Jacek; Wang, Xia; Lopez, Irma; Ren, Huanan; Chen, Yiyun; Li, Yumei; Fishman, Gerald A; Genead, Mohammed; Schwartzentruber, Jeremy; Solanki, Naimesh; Traboulsi, Elias I; Cheng, Jingliang; Logan, Clare V; McKibbin, Martin; Hayward, Bruce E; Parry, David A; Johnson, Colin A; Nageeb, Mohammed; Poulter, James A; Mohamed, Moin D; Jafri, Hussain; Rashid, Yasmin; Taylor, Graham R; Keser, Vafa; Mardon, Graeme; Xu, Huidan; Inglehearn, Chris F; Fu, Qing; Toomes, Carmel; Chen, Rui

    2012-09-01

    Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wld(s)) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder. PMID:22842230

  5. Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration

    PubMed Central

    Koenekoop, Robert K.; Wang, Hui; Majewski, Jacek; Wang, Xia; Lopez, Irma; Ren, Huanan; Chen, Yiyun; Li, Yumei; Fishman, Gerald A.; Genead, Mohammed; Schwartzentruber, Jeremy; Solanki, Naimesh; Traboulsi, Elias I.; Cheng, Jingliang; Logan, Clare V.; McKibbin, Martin; Hayward, Bruce E.; Parry, David A.; Johnson, Colin A.; Nageeb, Mohammed; Poulter, James A.; Mohamed, Moin D.; Jafri, Hussain; Rashid, Yasmin; Taylor, Graham R.; Keser, Vafa; Mardon, Graeme; Xu, Huidan; Inglehearn, Chris F.; Fu, Qing; Toomes, Carmel; Chen, Rui

    2013-01-01

    Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wlds) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder. PMID:22842230

  6. 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

  7. 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-Cladière, Laurence; Delage, Ludovic; Leroux, Cédric J.-J.; Goulitquer, Sophie; Leblanc, Catherine; Creis, Emeline; Gall, Erwan Ar; Stiger-Pouvreau, Valérie; 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-Å 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

  8. 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.; O’Toole, 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

  9. MANORAA (Mapping Analogous Nuclei Onto Residue And Affinity) for identifying protein–ligand fragment interaction, pathways and SNPs

    PubMed Central

    Tanramluk, Duangrudee; Narupiyakul, Lalita; Akavipat, Ruj; Gong, Sungsam; Charoensawan, Varodom

    2016-01-01

    Protein–ligand interaction analysis is an important step of drug design and protein engineering in order to predict the binding affinity and selectivity between ligands to the target proteins. To date, there are more than 100 000 structures available in the Protein Data Bank (PDB), of which ∼30% are protein–ligand (MW below 1000 Da) complexes. We have developed the integrative web server MANORAA (Mapping Analogous Nuclei Onto Residue And Affinity) with the aim of providing a user-friendly web interface to assist structural study and design of protein–ligand interactions. In brief, the server allows the users to input the chemical fragments and present all the unique molecular interactions to the target proteins with available three-dimensional structures in the PDB. The users can also link the ligands of interest to assess possible off-target proteins, human variants and pathway information using our all-in-one integrated tools. Taken together, we envisage that the server will facilitate and improve the study of protein–ligand interactions by allowing observation and comparison of ligand interactions with multiple proteins at the same time. (http://manoraa.org). PMID:27131358

  10. Gene Co-Expression Network Analysis for Identifying Modules and Functionally Enriched Pathways in Type 1 Diabetes.

    PubMed

    Riquelme Medina, Ignacio; Lubovac-Pilav, Zelmina

    2016-01-01

    Type 1 diabetes (T1D) is a complex disease, caused by the autoimmune destruction of the insulin producing pancreatic beta cells, resulting in the body's inability to produce insulin. While great efforts have been put into understanding the genetic and environmental factors that contribute to the etiology of the disease, the exact molecular mechanisms are still largely unknown. T1D is a heterogeneous disease, and previous research in this field is mainly focused on the analysis of single genes, or using traditional gene expression profiling, which generally does not reveal the functional context of a gene associated with a complex disorder. However, network-based analysis does take into account the interactions between the diabetes specific genes or proteins and contributes to new knowledge about disease modules, which in turn can be used for identification of potential new biomarkers for T1D. In this study, we analyzed public microarray data of T1D patients and healthy controls by applying a systems biology approach that combines network-based Weighted Gene Co-Expression Network Analysis (WGCNA) with functional enrichment analysis. Novel co-expression gene network modules associated with T1D were elucidated, which in turn provided a basis for the identification of potential pathways and biomarker genes that may be involved in development of T1D. PMID:27257970

  11. MANORAA (Mapping Analogous Nuclei Onto Residue And Affinity) for identifying protein-ligand fragment interaction, pathways and SNPs.

    PubMed

    Tanramluk, Duangrudee; Narupiyakul, Lalita; Akavipat, Ruj; Gong, Sungsam; Charoensawan, Varodom

    2016-07-01

    Protein-ligand interaction analysis is an important step of drug design and protein engineering in order to predict the binding affinity and selectivity between ligands to the target proteins. To date, there are more than 100 000 structures available in the Protein Data Bank (PDB), of which ∼30% are protein-ligand (MW below 1000 Da) complexes. We have developed the integrative web server MANORAA (Mapping Analogous Nuclei Onto Residue And Affinity) with the aim of providing a user-friendly web interface to assist structural study and design of protein-ligand interactions. In brief, the server allows the users to input the chemical fragments and present all the unique molecular interactions to the target proteins with available three-dimensional structures in the PDB. The users can also link the ligands of interest to assess possible off-target proteins, human variants and pathway information using our all-in-one integrated tools. Taken together, we envisage that the server will facilitate and improve the study of protein-ligand interactions by allowing observation and comparison of ligand interactions with multiple proteins at the same time. (http://manoraa.org). PMID:27131358

  12. Gene Co-Expression Network Analysis for Identifying Modules and Functionally Enriched Pathways in Type 1 Diabetes

    PubMed Central

    Riquelme Medina, Ignacio; Lubovac-Pilav, Zelmina

    2016-01-01

    Type 1 diabetes (T1D) is a complex disease, caused by the autoimmune destruction of the insulin producing pancreatic beta cells, resulting in the body’s inability to produce insulin. While great efforts have been put into understanding the genetic and environmental factors that contribute to the etiology of the disease, the exact molecular mechanisms are still largely unknown. T1D is a heterogeneous disease, and previous research in this field is mainly focused on the analysis of single genes, or using traditional gene expression profiling, which generally does not reveal the functional context of a gene associated with a complex disorder. However, network-based analysis does take into account the interactions between the diabetes specific genes or proteins and contributes to new knowledge about disease modules, which in turn can be used for identification of potential new biomarkers for T1D. In this study, we analyzed public microarray data of T1D patients and healthy controls by applying a systems biology approach that combines network-based Weighted Gene Co-Expression Network Analysis (WGCNA) with functional enrichment analysis. Novel co-expression gene network modules associated with T1D were elucidated, which in turn provided a basis for the identification of potential pathways and biomarker genes that may be involved in development of T1D. PMID:27257970

  13. Periodic Nanomechanical Stimulation in a Biokinetics Model Identifying Anabolic and Catabolic Pathways Associated With Cartilage Matrix Homeostasis

    PubMed Central

    Saha, Asit K.

    2010-01-01

    Enhancing the available nanotechnology to describe physicochemical interactions during biokinetic regulation will strongly support cellular and molecular engineering efforts. In a recent mathematical model developed to extend the applicability of a statically loaded, single-cell biomechanical analysis, a biokinetic regulatory threshold was presented (Saha and Kohles, 2010, “A Distinct Catabolic to Anabolic Threshold Due to Single-Cell Static Nanomechanical Stimulation in a Cartilage Biokinetics Model,” J. Nanotechnol. Eng. Med., 1(3), p. 031005). Results described multiscale mechanobiology in terms of catabolic to anabolic pathways. In the present study, we expand the mathematical model to continue exploring the nanoscale biomolecular response within a controlled microenvironment. Here, we introduce a dynamic mechanical stimulus for regulating cartilage molecule synthesis. Model iterations indicate the identification of a biomathematical mechanism balancing the harmony between catabolic and anabolic states. Relative load limits were defined to distinguish between “healthy” and “injurious” biomolecule accumulations. The presented mathematical framework provides a specific algorithm from which to explore biokinetic regulation. PMID:21152382

  14. 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

  15. Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

    PubMed Central

    2012-01-01

    Introduction Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease with complex etiopathogenesis. Despite extensive studies to understand the disease process utilizing human and mouse models, the intersection between these species remains elusive. To address this gap, we utilized a novel systems biology approach to identify disease-related gene modules and signaling pathways that overlap between humans and mice. Methods Parotid gland tissues were harvested from 24 pSS and 16 non-pSS sicca patients and 25 controls. For mouse studies, salivary glands were harvested from C57BL/6.NOD-Aec1Aec2 mice at various times during development of pSS-like disease. RNA was analyzed with Affymetrix HG U133+2.0 arrays for human samples and with MOE430+2.0 arrays for mouse samples. The images were processed with Affymetrix software. Weighted-gene co-expression network analysis was used to identify disease-related and functional pathways. Results Nineteen co-expression modules were identified in human parotid tissue, of which four were significantly upregulated and three were downregulated in pSS patients compared with non-pSS sicca patients and controls. Notably, one of the human disease-related modules was highly preserved in the mouse model, and was enriched with genes involved in immune and inflammatory responses. Further comparison between these two species led to the identification of genes associated with leukocyte recruitment and germinal center formation. Conclusion Our systems biology analysis of genome-wide expression data from salivary gland tissue of pSS patients and from a pSS mouse model identified common dysregulated biological pathways and molecular targets underlying critical molecular alterations in pSS pathogenesis. PMID:23116360

  16. Beneficial impact of intracerebroventricular fractalkine administration on behavioral and biochemical changes induced by prenatal stress in adult rats: Possible role of NLRP3 inflammasome pathway.

    PubMed

    Ślusarczyk, Joanna; Trojan, Ewa; Wydra, Karolina; Głombik, Katarzyna; Chamera, Katarzyna; Kucharczyk, Mateusz; Budziszewska, Bogusława; Kubera, Marta; Lasoń, Władysław; Filip, Małgorzata; Basta-Kaim, Agnieszka

    2016-08-01

    Several lines of evidence indicate that adverse experience in early life may be a triggering factor for pathological inflammatory processes and lead to the development of depression. Fractalkine (CX3CL1), a chemokine, plays an important role not only in the migration, differentiation and proliferation of neuronal and glial cells but also in the regulation of neuronal-microglial signaling and the production of pro-inflammatory factors. In the present study, we examined the impact of a prenatal stress procedure on the expression of fractalkine in the hippocampus and frontal cortex of young and adult male rats. Furthermore, we measured the age-dependent effect of stress during pregnancy on the expression of pro-inflammatory factors IL-1β, IL-18, TNF-α, IL-6, and CCL2 in both brain structures. Next, to illustrate the link between fractalkine signaling and the behavioral and biochemical changes induced by prenatal stress, adult prenatally stressed offspring were injected intracerebroventricularly (icv) with exogenous fractalkine. We reported that prenatal stress leads to long-lasting deficits in fractalkine signaling and enhanced inflammatory activation. The study demonstrates that icv administration of fractalkine attenuates the behavioural changes evoked by prenatal stress procedure in adult animals. Moreover, fractalkine administration, exhibits anti-inflammatory action, mainly in the frontal cortex of adult prenatally stressed rats. The effect of fractalkine is related to inhibition of NLRP3 inflammasome. However, its action on the other members of NOD-like receptor family (NLR) cannot be excluded. These findings provide new in vivo evidence that the behavioral and inflammatory disturbances observed in adult prenatally stressed rats may be related to long-lasting malfunctions in fractalkine signaling. PMID:27206338

  17. Identifying resistance mechanisms against five tyrosine kinase inhibitors targeting the ERBB/RAS pathway in 45 cancer cell lines.

    PubMed

    Pénzváltó, Zsófia; Tegze, Bálint; Szász, A Marcell; Sztupinszki, Zsófia; Likó, István; Szendrői, Attila; Schäfer, Reinhold; Győrffy, Balázs

    2013-01-01

    Because of the low overall response rates of 10-47% 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. 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

  19. Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease

    PubMed Central

    Zawada, Adam M; Rogacev, Kyrill S; Müller, Sören; Rotter, Björn; Winter, Peter; Fliser, Danilo; Heine, Gunnar H

    2014-01-01

    Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD. PMID:24184689

  20. Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease.

    PubMed

    Zawada, Adam M; Rogacev, Kyrill S; Müller, Sören; Rotter, Björn; Winter, Peter; Fliser, Danilo; Heine, Gunnar H

    2014-01-01

    Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD. PMID:24184689

  1. Multigene pathway-based analyses identify nasopharyngeal carcinoma risk associations for cumulative adverse effects of TERT-CLPTM1L and DNA double-strand breaks repair.

    PubMed

    Yee Ko, Josephine Mun; Dai, Wei; Wun Wong, Elibe Hiu; Kwong, Dora; Tong Ng, Wai; Lee, Anne; Kai Cheong Ngan, Roger; Chung Yau, Chun; Tung, Stewart; Li Lung, Maria

    2014-10-01

    The genetic etiology of nasopharyngeal carcinoma (NPC) and mechanisms for inherited susceptibility remain unclear. To examine genetic risk factors for NPC, we hypothesized that heritable risk is attributable to cumulative effects of multiple common low-risk variants. With the premise that individual SNPs only confer subtle effects for cancer risk, a multigenic pathway-based approach was used to systematically examine associations between NPC genetic susceptibility with SNPs in genes in DNA repair pathways and from previously identified cancer genome-wide association study analyses. This case-control study covers 161 genes/loci and focuses on pathway-based analyses in 2,349 Hong Kong individuals, allowing stratification according to NPC familial status for meaningful association analysis. Three SNPs (rs401681, rs6774494 and rs3757318) corresponding to TERT/CLPTM1L (OR 95% CI = 0.77, 0.68-0.88), MDS1-EVI1 (OR 95% CI=0.79 0.69-0.89) and CCDC170 (OR 95% CI = 0.76, 0.66-0.86) conferred modest protective effects individually for NPC risk by the logistic regression analysis after multiple testing adjustment (p(Bonferroni)  < 0.05). Stratification of NPC according to familial status identified rs2380165 in BLM (OR 95% CI = 1.49, 1.20-1.86, p(Bonferroni)  < 0.05) association with family history-positive NPC (FH+ NPC) patients. Multiple SNPs pathway-based analysis revealed that the combined gene dosage effects for increasing numbers of unfavorable genotypes in TERT-CLPTM1L and double-strand break repair (DSBR) conferred elevated risk in FH+ and sporadic NPC patients (ORs per allele, 95% CIs = 1.37, 1.22-1.55, p(Bonferroni)  = 5.00 × 10(-6); 1.17, 1.09-1.26, p(Bonferroni)  = 4.58 × 10(-4) , respectively, in TERT/NHEJ pathways). Our data suggested cumulative increased NPC risk associations with TERT-CLPTM1L and DSBR pathways contribute to genetic susceptibility to NPC and have potential translational relevance for patient stratification and therapeutics. PMID

  2. Secretome Analysis Identifies Novel Signal Peptide Peptidase-Like 3 (SPPL3) Substrates and Reveals a Role of SPPL3 in Multiple Golgi Glycosylation Pathways*

    PubMed Central

    Kuhn, Peer-Hendrik; Voss, Matthias; Haug-Kröper, Martina; Schröder, Bernd; Schepers, Ute; Bräse, Stefan; Haass, Christian; Lichtenthaler, Stefan F.; Fluhrer, Regina

    2015-01-01

    Signal peptide peptidase-like 3 (SPPL3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, SPPL3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological SPPL3 substrates. SPPL3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are SPPL3 substrates or whether SPPL3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify SPPL3 substrates we used Sppl3-deficient and SPPL3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics “secretome protein enrichment with click sugars (SPECS)” method. SPECS analysis identified numerous additional new SPPL3 candidate glycoprotein substrates, several of which were biochemically validated as SPPL3 substrates. All novel SPPL3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel SPPL3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that SPPL3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, SPPL3 emerges as a crucial player of Golgi function and the newly identified SPPL3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of SPPL3 in the Golgi network and in vivo

  3. 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

  4. 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-κB inhibitor ɛ (IκBɛ), 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

  5. Hippocampal Gene Expression Meta-Analysis Identifies Aging and Age-Associated Spatial Learning Impairment (ASLI) Genes and Pathways

    PubMed Central

    Uddin, Raihan K.; Singh, Shiva M.

    2013-01-01

    A number of gene expression microarray studies have been carried out in the past, which studied aging and age-associated spatial learning impairment (ASLI) in the hippocampus in animal models, with varying results. Data from such studies were never integrated to identify the most significant ASLI genes and to understand their effect. In this study we integrated these data involving rats using meta-analysis. Our results show that proper removal of batch effects from microarray data generated from different laboratories is necessary before integrating them for meta-analysis. Our meta-analysis has identified a number of significant differentially expressed genes across age or across ASLI. These genes affect many key functions in the aged compared to the young rats, which include viability of neurons, cell-to-cell signalling and interaction, migration of cells, neuronal growth, and synaptic plasticity. These functional changes due to the altered gene expression may manifest into various neurodegenerative diseases and disorders, some of which leading into syndromic memory impairments. While other aging related molecular changes can result into altered synaptic plasticity simply causing normal aging related non-syndromic learning or spatial learning impairments such as ASLI. PMID:23874995

  6. Gene Expression Profiling Identifies Cell Proliferation and Inflammation as the Predominant Pathways Regulated by Aryl Hydrocarbon Receptor in Primary Human Fetal Lung Cells Exposed to Hyperoxia.

    PubMed

    Shivanna, Binoy; Maity, Suman; Zhang, Shaojie; Patel, Ananddeep; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E; Belmont, John; Coarfa, Cristian; Moorthy, Bhagavatula

    2016-07-01

    Exposure to hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. We observed that aryl hydrocarbon receptor (AhR) signaling protects newborn mice and primary fetal human pulmonary microvascular endothelial cells (HPMECs) against hyperoxic injury. Additionally, a recent genome-wide transcriptome study in a newborn mouse model of BPD identified AhR as a key regulator of hyperoxia-induced gene dysregulation. Whether the AhR similarly deregulates genes in HPMEC is unknown. Therefore, the objective of this study was to characterize transcriptome level gene expression profile in AhR-sufficient and -deficient HPMEC exposed to normoxic and hyperoxic conditions. Global gene expression profiling was performed using Illumina microarray platform and selected genes were validated by real-time RT-PCR. AhR gene expression and hyperoxia independently affected the expression of 540 and 593 genes, respectively. Two-way ANOVA further identified 85 genes that were affected by an interaction between AhR expression and exposure to hyperoxia. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology, and Reactome pathway analysis identified cell proliferation, immune function, cytokine signaling, and organ development as the major pathways affected in AhR-deficient cells. The biological processes that were significantly enriched by hyperoxia included metabolic process, stress response, signal transduction, cell cycle, and immune regulation. Cell cycle was the predominant pathway affected by the combined effect of AhR knockdown and hyperoxia. Functional analysis of cell cycle showed that AhR-deficient cells had decreased proliferation compared with AhR-sufficient cells. These findings suggest that AhR modulates hyperoxic lung injury by regulating the genes that are necessary for cell proliferation and inflammation. PMID:27103661

  7. Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways.

    PubMed

    Scott, Robert A; Lagou, Vasiliki; Welch, Ryan P; Wheeler, Eleanor; Montasser, May E; Luan, Jian'an; Mägi, Reedik; Strawbridge, Rona J; Rehnberg, Emil; Gustafsson, Stefan; Kanoni, Stavroula; Rasmussen-Torvik, Laura J; Yengo, Loïc; 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; O'Connell, Jeffrey R; Stirrups, Kathleen; Vitart, Veronique; Hayward, Caroline; Esko, Tõnu; 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; Müller-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, Göran; 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; Lindström, 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; Körner, 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, Josée; 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, Inês

    2012-09-01

    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 increased 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 concentration 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 analysis of these newly discovered loci will further improve our understanding of glycemic control. PMID:22885924

  8. 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, Jian’an; Mägi, Reedik; Strawbridge, Rona J; Rehnberg, Emil; Gustafsson, Stefan; Kanoni, Stavroula; Rasmussen-Torvik, Laura J; Yengo, Loïc; 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; O’Connell, Jeffrey R; Stirrups, Kathleen; Vitart, Veronique; Hayward, Caroline; Esko, Tönu; 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; Müller-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, Göran; 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; Lindström, 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; Körner, 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, Josée; 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, Inês

    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

  9. Multiple biomarker tissue arrays: A computational approach to identifying protein-protein interactions in the EGFR/ERK signalling pathway

    PubMed Central

    2012-01-01

    Background Many studies have demonstrated genetic and environmental factors that lead to renal cell carcinoma (RCC) and that occur during a protracted period of tumourigenesis. It appears suitable to identify and characterise potential molecular markers that appear during tumourigenesis and that might provide rapid and effective possibilities for the early detection of RCC. EGFR activation induces cell cycle progression, inhibition of apoptosis and angiogenesis, promotion of invasion/metastasis, and other tumour promoting activities. Over-expression of EGFR is thought to play an important role in tumour initiation and progression of RCC because up-regulation of EGFR has been associated with high grade cancers and a worse prognosis. Methods Characterisation of the protein profile interacting with EGFR was performed using the following: an immunohistochemical (IHC) study of EGFR, a comprehensive computational study of EGFR protein-protein interactions, an analysis correlating the expression levels of EGFR with other significant markers in the tumourigenicity of RCC, and finally, an analysis of the utility of EGFR for prognosis in a cohort of patients with renal cell carcinoma. Results The cases that showed a higher level of this protein fell within the clear cell histological subtype (p = 0.001). The EGFR significance statistic was found with respect to a worse prognosis. In vivo significant correlations were found with PDGFR-β, Flk-1, Hif1-α, proteins related to differentiation (such as DLL3 and DLL4 ligands), and certain metabolic proteins such as Glut5. In silico significant associations gave us a panel of 32 EGFR-interacting proteins (EIP) using the APID and STRING databases. Conclusions This work summarises the multifaceted role of EGFR in the pathology of RCC, and it identifies EIPs that could help to provide mechanistic explanations for the different behaviours observed in tumours. PMID:22937740

  10. 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

  11. Arachidonic Acid Pathway Members PLA2G7, HPGD, EPHX2, and CYP4F8 Identified as Putative Novel Therapeutic Targets in Prostate Cancer

    PubMed Central

    Vainio, Paula; Gupta, Santosh; Ketola, Kirsi; Mirtti, Tuomas; Mpindi, John-Patrick; Kohonen, Pekka; Fey, Vidal; Perälä, Merja; Smit, Frank; Verhaegh, Gerald; Schalken, Jack; Alanen, Kalle A.; Kallioniemi, Olli; Iljin, Kristiina

    2011-01-01

    The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress. PMID:21281786

  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. Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice.

    PubMed

    Tkatchenko, Andrei V; Luo, Xiaoyan; Tkatchenko, Tatiana V; Vaz, Candida; Tanavde, Vivek M; Maurer-Stroh, Sebastian; Zauscher, Stefan; Gonzalez, Pedro; Young, Terri L

    2016-01-01

    Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlighted that changes in microRNA expression are involved in the regulation of refractive eye development and predicted how they may be involved in the development of myopia by regulating retinal gene expression. PMID:27622715

  14. Quantitation of Aristolochic Acids in Corn, Wheat Grain, and Soil Samples Collected in Serbia: Identifying a Novel Exposure Pathway in the Etiology of Balkan Endemic Nephropathy.

    PubMed

    Chan, Wan; Pavlović, Nikola M; Li, Weiwei; Chan, Chi-Kong; Liu, Jingjing; Deng, Kailin; Wang, Yinan; Milosavljević, Biljana; Kostić, Emina N

    2016-07-27

    While to date investigations provided convincing evidence on the role of aristolochic acids (AAs) in the etiology of Balkan endemic nephropathy (BEN) and upper urothelial cancer (UUC), the exposure pathways by which AAs enter human bodies to cause BEN and UUC remain obscure. The goal of this study is to test the hypothesis that environmental pollution by AAs and root uptake of AAs in the polluted soil may be one of the pathways by which AAs enter the human food chain. The hypothesis driving this study was that the decay of Aristolochia clematitis L., a AA-containing herbaceous plant that is found growing widespread in the endemic regions, could release free AAs to the soil, which could be taken up by food crops growing nearby, thereby transferring this potent human nephrotoxin and carcinogen into their edible parts. Using the highly sensitive and selective high-performance liquid chromatography coupled with fluorescence detection method, we identified and quantitated in this study for the first time AAs in corn, wheat grain, and soil samples collected from the endemic village Kutles in Serbia. Our results provide the first direct evidence that food crops and soil in the Balkans are contaminated with AAs. It is possible that the presence of AAs in edible parts of crops originating from the AA-contaminated soil could be one of the major pathways by which humans become exposed to AAs. PMID:27362729

  15. 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

  16. An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway

    PubMed Central

    Gonsalves, Foster C.; Klein, Keren; Carson, Brittany B.; Katz, Shauna; Ekas, Laura A.; Evans, Steve; Nagourney, Robert; Cardozo, Timothy; Brown, Anthony M. C.; DasGupta, Ramanuj

    2011-01-01

    Misregulated β-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of β-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear β-catenin. We show that these inhibitors efficiently block Wnt/β-catenin–induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling. PMID:21393571

  17. The Nematode Caenorhabditis elegans, Stress and Aging: Identifying the Complex Interplay of Genetic Pathways Following the Treatment with Humic Substances

    PubMed Central

    Menzel, Ralph; Menzel, Stefanie; Swain, Suresh C.; Pietsch, Kerstin; Tiedt, Sophie; Witczak, Jördis; Stürzenbaum, Stephen R.; Steinberg, Christian E. W.

    2012-01-01

    Low concentrations of the dissolved leonardite humic acid HuminFeed® (HF) prolonged the lifespan and enhanced the thermal stress resistance of the model organism Caenorhabditis elegans. However, growth was impaired and reproduction delayed, effects which have also been identified in response to other polyphenolic monomers, including Tannic acid, Rosmarinic acid, and Caffeic acid. Moreover, a chemical modification of HF, which increases its phenolic/quinonoid moieties, magnified the biological impact on C. elegans. To gain a deep insight into the molecular basis of these effects, we performed global transcriptomics on young adult (3 days) and old adult (11 days) nematodes exposed to two different concentrations of HF. We also studied several C. elegans mutant strains in respect to HF derived longevity and compared all results with data obtained for the chemically modified HF. The gene expression pattern of young HF-treated nematodes displayed a significant overlap to other conditions known to provoke longevity, including various plant polyphenol monomers. Besides the regulation of parts of the metabolism, transforming growth factor-beta signaling, and Insulin-like signaling, lysosomal activities seem to contribute most to HF’s and modified HF’s lifespan prolonging action. These results support the notion that the phenolic/quinonoid moieties of humic substances are major building blocks that drive the physiological effects observed in C. elegans. PMID:22529848

  18. The Nematode Caenorhabditis elegans, Stress and Aging: Identifying the Complex Interplay of Genetic Pathways Following the Treatment with Humic Substances.

    PubMed

    Menzel, Ralph; Menzel, Stefanie; Swain, Suresh C; Pietsch, Kerstin; Tiedt, Sophie; Witczak, Jördis; Stürzenbaum, Stephen R; Steinberg, Christian E W

    2012-01-01

    Low concentrations of the dissolved leonardite humic acid HuminFeed(®) (HF) prolonged the lifespan and enhanced the thermal stress resistance of the model organism Caenorhabditis elegans. However, growth was impaired and reproduction delayed, effects which have also been identified in response to other polyphenolic monomers, including Tannic acid, Rosmarinic acid, and Caffeic acid. Moreover, a chemical modification of HF, which increases its phenolic/quinonoid moieties, magnified the biological impact on C. elegans. To gain a deep insight into the molecular basis of these effects, we performed global transcriptomics on young adult (3 days) and old adult (11 days) nematodes exposed to two different concentrations of HF. We also studied several C. elegans mutant strains in respect to HF derived longevity and compared all results with data obtained for the chemically modified HF. The gene expression pattern of young HF-treated nematodes displayed a significant overlap to other conditions known to provoke longevity, including various plant polyphenol monomers. Besides the regulation of parts of the metabolism, transforming growth factor-beta signaling, and Insulin-like signaling, lysosomal activities seem to contribute most to HF's and modified HF's lifespan prolonging action. These results support the notion that the phenolic/quinonoid moieties of humic substances are major building blocks that drive the physiological effects observed in C. elegans. PMID:22529848

  19. YlxM is a newly identified accessory protein that influences the function of signal recognition particle pathway components in Streptococcus mutans.

    PubMed

    Williams, Matthew L; Crowley, Paula J; Hasona, Adnan; Brady, L Jeannine

    2014-06-01

    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

  20. 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

  1. Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

    PubMed

    Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

    2014-05-01

    The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes. PMID:24817326

  2. 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

  3. A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.

    PubMed

    Voter, Andrew F; Manthei, Kelly A; Keck, James L

    2016-07-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol. PMID:26962873

  4. 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.

  5. CSGene: a literature-based database for cell senescence genes and its application to identify critical cell aging pathways and associated diseases

    PubMed Central

    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

  6. 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...

  7. 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...

  8. 40 CFR 158.2010 - Biochemical pesticides data requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 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, 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...

  10. 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...

  11. Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response

    PubMed Central

    Richard, Alexandra; Corvol, Jean-Christophe; Debs, Rabab; Reach, Pauline; Tahiri, Khadija; Carpentier, Wassila; Gueguen, Justine; Guillemot, Vincent; Labeyrie, Céline; Adams, David; Viala, Karine; Cohen Aubart, Fleur

    2016-01-01

    Abstract We have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83–6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-α receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-α-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-α gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity

  12. Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response.

    PubMed

    Richard, Alexandra; Corvol, Jean-Christophe; Debs, Rabab; Reach, Pauline; Tahiri, Khadija; Carpentier, Wassila; Gueguen, Justine; Guillemot, Vincent; Labeyrie, Céline; Adams, David; Viala, Karine; Cohen Aubart, Fleur

    2016-05-01

    We have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83-6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-α receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-α-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-α gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity that is

  13. 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

  14. Robust Selection Algorithm (RSA) for Multi-Omic Biomarker Discovery; Integration with Functional Network Analysis to Identify miRNA Regulated Pathways in Multiple Cancers.

    PubMed

    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

  15. Genomic and Immunological Tumor Profiling Identifies Targetable Pathways and Extensive CD8+/PDL1+ Immune Infiltration in Inflammatory Breast Cancer Tumors.

    PubMed

    Hamm, Christopher A; Moran, Diarmuid; Rao, Kakuturu; Trusk, Patricia B; Pry, Karen; Sausen, Mark; Jones, Siân; Velculescu, Victor E; Cristofanilli, Massimo; Bacus, Sarah

    2016-07-01

    Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that remains poorly understood at the molecular level. Comprehensive tumor profiling was performed to understand clinically actionable alterations in IBC. Targeted next-generation sequencing (NGS) and IHC were performed to identify activated pathways in IBC tumor tissues. siRNA studies examined the impact of IBC genomic variants in cellular models. IBC tumor tissues were further characterized for immune infiltration and immune checkpoint expression by IHC. Genomic analysis identified recurrent alterations in core biologic pathways, including activating and targetable variants in HER/PI3K/mTOR signaling. High rates of activating HER3 point mutations were discovered in IBC tumors. Cell line studies confirmed a role for mutant HER3 in IBC cell proliferation. Immunologic analysis revealed a subset of IBC tumors associated with high CD8(+)/PD-L1(+) lymphocyte infiltration. Immune infiltration positively correlated with an NGS-based estimate of neoantigen exposure derived from the somatic mutation rate and mutant allele frequency, iScore. Additionally, DNA mismatch repair alterations, which may contribute to higher iScores, occurred at greater frequency in tumors with higher immune infiltration. Our study identifies genomic alterations that mechanistically contribute to oncogenic signaling in IBC and provides a genetic basis for the selection of clinically relevant targeted and combination therapeutic strategies. Furthermore, an NGS-based estimate of neoantigen exposure developed in this study (iScore) may be a useful biomarker to predict immune infiltration in IBC and other cancers. The iScore may be associated with greater levels of response to immunotherapies, such as PD-L1/PD-1-targeted therapies. Mol Cancer Ther; 15(7); 1746-56. ©2016 AACR. PMID:27196778

  16. Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses*

    PubMed Central

    Matsumura, Satoko; Shinoda, Keiko; Yamada, Mayumi; Yokojima, Satoshi; Inoue, Masafumi; Ohnishi, Takayuki; Shimada, Tetsuya; Kikuchi, Kazuya; Masui, Dai; Hashimoto, Shigeki; Sato, Michio; Ito, Akane; Akioka, Manami; Takagi, Shinsuke; Nakamura, Yoshihiro; Nemoto, Kiyokazu; Hasegawa, Yutaka; Takamoto, Hisayoshi; Inoue, Haruo; Nakamura, Shinichiro; Nabeshima, Yo-ichi; Teplow, David B.; Kinjo, Masataka; Hoshi, Minako

    2011-01-01

    Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys16, we uncovered two distinct assembly pathways. One leads to highly toxic 10–15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15–40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys16-labeled peptide disturbed fibril formation because the Aβ16–20 region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial. PMID:21292768

  17. Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFβR3 In Vitro

    PubMed Central

    DeLaughter, Daniel M.; Clark, Cynthia R.; Christodoulou, Danos C.; Seidman, Christine E.; Baldwin, H. Scott; Seidman, J. G.; Barnett, Joey V.

    2016-01-01

    The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFβR3 (TGFβ1, TGFβ2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFβ1 (515), TGFβ2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFβ1-TGFβ2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFβ and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFβ2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFβ2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFβR3. Overall, the genes and signaling pathways identified

  18. Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A.

    PubMed

    Pereira, Maria da Conceição; Morais, Sara; Sequeiros, Jorge; Alonso, Isabel

    2016-04-01

    Variants inCACNA1Athat encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca(2+)channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using theCaenorhabditis elegansstrain CB55, which carries a truncating mutation in theunc-2gene, the worm ortholog for the humanCACNA1A The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype ofunc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in theC. elegansneurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related tounc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway. PMID:27005779

  19. Time point-based integrative analyses of deep-transcriptome identify four signal pathways in blastemal regeneration of zebrafish lower jaw.

    PubMed

    Zhang, Hui; Wang, Xuelong; Lyu, Kailun; Gao, Siqi; Wang, Guan; Fan, Chunxin; Zhang, Xin A; Yan, Jizhou

    2015-03-01

    There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. We have previously reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, re-epitheliation, blastemal formation, and respecification. By combining the hierarchical gene ontology term network, the DAVID annotation system, and Euclidean distance clustering, we identified four signaling pathways: foxi1-foxo1b-pou3f1, pax3a-mant3a-col11/col2, pou5f1-cdx4-kdrl, and isl1-wnt11 PCP-sox9a. Results from immunohistochemical staining and promoter-driven transgenic fish suggest that these pathways, respectively, define wound epidermis reconstitution, cell type conversions, blastemal angiogenesis/vasculogenesis, and cartilage matrix-orientation. Foxi1 morpholino-knockdown caused expansions of Foxo1b- and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9a and hoxa2b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including re-epitheliation and blastema formation. This study provides novel insight into a blastema regeneration strategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition. PMID:25420467

  20. 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.

  1. Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A

    PubMed Central

    Pereira, Maria da Conceição; Morais, Sara; Sequeiros, Jorge

    2016-01-01

    Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway. PMID:27005779

  2. 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

  3. 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

  4. Candidate Agtr2 influenced genes and pathways identified by expression profiling in the developing brain of Agtr2−/y mice

    PubMed Central

    Pawlowski, Traci L.; Heringer-Walther, Silvia; Cheng, Chun-Huai; Archie, John G.; Chen, Chin-Fu; Walther, Thomas; Srivastava, Anand K.

    2009-01-01

    Intellectual disability (ID) is a common developmental disability observed in one to three percent of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2−/y) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2−/y and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2−/y brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p ≤ 0.005) at E15 and in P1 brains of the Agtr2−/y mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory. PMID:19501643

  5. 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, André 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

  6. 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

  7. Systematic Review of Micro-RNA Expression in Pre-Eclampsia Identifies a Number of Common Pathways Associated with the Disease

    PubMed Central

    Sheikh, Adam M.; Currie, Gemma; Delles, Christian

    2016-01-01

    Background Pre-eclampsia (PE) is a complex, multi-systemic condition of pregnancy which greatly impacts maternal and perinatal morbidity and mortality. MicroRNAs (miRs) are differentially expressed in PE and may be important in helping to understand the condition and its pathogenesis. Methods Case-control studies investigating expression of miRs in PE were collected through a systematic literature search. Data was extracted and compared from 58 studies to identify the most promising miRs associated with PE pathogenesis and identify areas of methodology which could account for often conflicting results. Results Some of the most frequently differentially expressed miRs in PE include miR-210, miR-223 and miR-126/126* which associate strongly with the etiological domains of hypoxia, immunology and angiogenesis. Members of the miR-515 family belonging to the imprinted chromosome 19 miR cluster with putative roles in trophoblast invasion were also found to be differentially expressed. Certain miRs appear to associate with more severe forms of PE such as miR-210 and the immune-related miR-181a and miR-15 families. Patterns of miR expression may help pinpoint key pathways (e.g. IL-6/miR-223/STAT3) and aid in untangling the heterogeneous nature of PE. The detectable presence of many PE-associated miRs in antenatal circulatory samples suggests their usefulness as predictive biomarkers. Further progress in ascertaining the clinical value of miRs and in understanding how they might contribute to pathogenesis is predicated upon resolving current methodological challenges in studies. These include differences in diagnostic criteria, cohort characteristics, sampling technique, RNA isolation and platform-dependent variation in miR profiling. Conclusion Reviewing studies of PE-associated miRs has revealed their potential as informants of underlying target genes and pathways relating to PE pathogenesis. However, the incongruity in results across current studies hampers their

  8. 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

  9. RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction.

    PubMed

    Bolton, Melvin D; Ebert, Malaika K; Faino, Luigi; Rivera-Varas, Viviana; de Jonge, Ronnie; Van de Peer, Yves; Thomma, Bart P H J; Secor, Gary A

    2016-07-01

    Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola. PMID:27112724

  10. 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

    Laurent, Audrey; Calabrese, Manuela; Warnatz, Hans-Jörg; 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

  11. 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

  12. Identifying early pathways of risk and resilience: The co-development of internalizing and externalizing symptoms and the role of harsh parenting

    PubMed Central

    Wiggins, Jillian Lee; Mitchell, Colter; Hyde, Luke W.; Monk, Christopher S.

    2016-01-01

    Psychological disorders co-occur often in children, but little has been done to document the types of conjoint pathways internalizing and externalizing symptoms may take from the crucial early period of toddlerhood or how harsh parenting may overlap with early symptom co-development. To examine symptom co-development trajectories, we identified latent classes of individuals based on internalizing and externalizing symptoms across ages 3–9 and found three symptom co-development classes: normative symptoms (low), severe-decreasing symptoms (initially high but rapidly declining) and severe symptoms (high) trajectories. Next, joint models examined how parenting trajectories overlapped with internalizing and externalizing symptom trajectories. These trajectory classes demonstrated that, normatively, harsh parenting increased after toddlerhood, but the severe symptoms class was characterized by a higher level and steeper increase in harsh parenting and the severe-decreasing class by high, stable harsh parenting. Additionally, a transactional model examined the bi-directional relationships among internalizing and externalizing symptoms and harsh parenting as they may cascade over time in this early period. Harsh parenting uniquely contributed to externalizing symptoms, controlling for internalizing symptoms, but not vice versa. Also, internalizing symptoms appeared to be a mechanism by which externalizing symptoms increase. Results highlight the importance accounting for both internalizing and externalizing symptoms from an early age to understand risk for developing psychopathology and the role harsh parenting plays in influencing these trajectories. PMID:26439075

  13. 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-Jörg; 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

  14. 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

  15. 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

  16. 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.

  17. Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis).

    PubMed

    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

  18. 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

  19. The Formation and Aerosol Uptake of Isoprene Nitrooxyhydroxyepoxide (INHE), a Newly Identified Product from the RO2 + HO2 Pathway of Isoprene NO3 Oxidation

    NASA Astrophysics Data System (ADS)

    Schwantes, R.; Teng, A.; Nguyen, T.; Coggon, M. M.; Zhang, X.; Schilling-Fahnestock, K.; Crounse, J.; St Clair, J. M.; Seinfeld, J.; Wennberg, P. O.

    2014-12-01

    Isoprene (C5H8) reacts with the nitrate radical (NO3) during the night to produce a peroxy nitrate radical (RO2). This RO2 can react with nitrogen oxides (i.e., NO, NO2, or NO3) and other RO2 radicals to form isoprene nitrates or with the hydroperoxyl radical (HO2) to form nitrooxyhydroperoxide (INP). Both model and field studies have found that in the ambient atmosphere much of the RO2 radical reacts with HO2. More specifically, during the 2013 SOAS field campaign, INP was one of the main species that increased at sunset suggesting the RO2 + HO2 pathway from NO3 oxidation is important in the southeastern US and similar areas. However, chamber studies so far have been run under conditions that optimize RO2 + NO3 reactions and/or RO2 + RO2 reactions. In this work, we present a new way to run NO3 oxidation chamber experiments that optimize for the RO2 + HO2 pathway creating a more atmospherically relevant product distribution. The gas phase formation of INP and subsequent oxidation products were monitored using a chemical ionization mass spectrometer (CIMS). Because isoprene nitrates formed from NO3 oxidation react slowly with ozone (O3) and NO3, many of these nitrates will remain in the atmosphere until the sun rises and hydroxyl radical (OH) begins to form. Results from these chamber experiments suggest that OH will react with INP to form nitrooxyhydroxyepoxide (INHE), a newly identified product from INP. We suspect INHE could be important for Secondary Organic Aerosol (SOA) production due to its similarity to isoprene epoxydiol (IEPOX), a product from isoprene OH oxidation that has been shown to be a significant SOA precursor. We studied the uptake of INHE onto various seed types, and found that as expected INHE rapidly partitions to highly acidic seed aerosol due to an acid catalyzed ring opening. A time-of-flight aerosol mass spectrometer (ToF-AMS) was used to understand the chemical composition of the aerosol produced from the various seed types.

  20. Comprehensive proteomic profiling identifies the androgen receptor axis and other signaling pathways as targets of microRNAs suppressed in metastatic prostate cancer.

    PubMed

    Coarfa, C; Fiskus, W; Eedunuri, V K; Rajapakshe, K; Foley, C; Chew, S A; Shah, S S; Geng, C; Shou, J; Mohamed, J S; O'Malley, B W; Mitsiades, N

    2016-05-01

    MicroRNAs are important epigenetic regulators of protein expression by triggering degradation of target mRNAs and/or inhibiting their translation. Dysregulation of microRNA expression has been reported in several cancers, including prostate cancer (PC). We comprehensively characterized the proteomic footprint of a panel of 12 microRNAs that are potently suppressed in metastatic PC (SiM-miRNAs: miR-1, miR-133a, miR-133b, miR-135a, miR-143-3p, miR-145-3p, miR-205, miR-221-3p, miR-221-5p, miR-222-3p, miR-24-1-5p, and miR-31) using reverse-phase proteomic arrays. Re-expression of these SiM-miRNAs in PC cells suppressed cell proliferation and targeted key oncogenic pathways, including cell cycle, apoptosis, Akt/mammalian target of rapamycin signaling, metastasis and the androgen receptor (AR) axis. However, only 12%, at most, of these observed protein expression changes could be explained by predicted direct binding of miRNAs to corresponding mRNAs, suggesting that the majority of these proteomic effects result indirectly. AR and its steroid receptor coactivators (SRCs; SRC-1, -2 and -3) were recurrently affected by these SiM-miRNAs. In agreement, we identified inverse correlations between expression of these SiM-miRNAs and early clinical recurrence, as well as with AR transcriptional activity in human PC tissues. We also identified robust induction of miR-135a by androgen and strong direct binding of AR to the miR-135a locus. As miR-135a potently suppresses AR expression, this results in a negative feedback loop that suppresses AR protein expression in an androgen-dependent manner, while de-repressing AR expression upon androgen deprivation. Our results demonstrate that epigenetic silencing of these SiM-miRNAs can result in increased AR axis activity and cell proliferation, thus contributing to disease progression. We further demonstrate that a negative feedback loop involving miR-135a can restore AR expression under androgen-deprivation conditions, thus contributing

  1. 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

  2. A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer

    PubMed Central

    Su, Bing; Gao, Lingqiu; Baranowski, Catherine; Gillard, Bryan; Wang, Jianmin; Ransom, Ryan; Ko, Hyun-Kyung; Gelman, Irwin H.

    2014-01-01

    Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness. PMID:24983969

  3. 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. ...

  4. 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

  5. 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

  6. 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

  7. 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 examples and for a more complex model of NF-κB pathway. Conclusion Our approach allows critical parameter identification and produces hierarchies of models. Hierarchical modeling is important in "middle-out" approaches when there is need to zoom in and out several levels of complexity. Critical parameter identification is an important issue in systems biology with potential applications to biological control and therapeutics. Our approach also deals naturally with the presence of multiple time scales, which is a general property of systems biology models. PMID:18854041

  8. Biochemical transformation of coals

    DOEpatents

    Lin, M.S.; Premuzic, E.T.

    1999-03-23

    A method of biochemically transforming macromolecular compounds found in solid carbonaceous materials, such as coal is provided. The preparation of new microorganisms, metabolically weaned through challenge growth processes to biochemically transform solid carbonaceous materials at extreme temperatures, pressures, pH, salt and toxic metal concentrations is also disclosed. 7 figs.

  9. 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

  10. Symptoms of major depressive disorder subsequent to child maltreatment: Examining change across multiple levels of analysis to identify transdiagnostic risk pathways.

    PubMed

    Shenk, Chad E; Griffin, Amanda M; O'Donnell, Kieran J

    2015-11-01

    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: neuroendocrine, autonomic, affective, and 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 18 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

  11. Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

    PubMed Central

    Mukherjee, Ali; Badal, Youssouf; Nguyen, Xuan-Thao; Miller, Johanna; Chenna, Ahmed; Tahir, Hasan; Newton, Alicia; Parry, Gordon; Williams, Stephen

    2011-01-01

    Background The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples. Methodology and Principal Findings Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation. Significance This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models. PMID:21297994

  12. The Fanconi anaemia pathway: new players and new functions.

    PubMed

    Ceccaldi, Raphael; Sarangi, Prabha; D'Andrea, Alan D

    2016-06-01

    The Fanconi anaemia pathway repairs DNA interstrand crosslinks (ICLs) in the genome. Our understanding of this complex pathway is still evolving, as new components continue to be identified and new biochemical systems are used to elucidate the molecular steps of repair. The Fanconi anaemia pathway uses components of other known DNA repair processes to achieve proper repair of ICLs. Moreover, Fanconi anaemia proteins have functions in genome maintenance beyond their canonical roles of repairing ICLs. Such functions include the stabilization of replication forks and the regulation of cytokinesis. Thus, Fanconi anaemia proteins are emerging as master regulators of genomic integrity that coordinate several repair processes. Here, we summarize our current understanding of the functions of the Fanconi anaemia pathway in ICL repair, together with an overview of its connections with other repair pathways and its emerging roles in genome maintenance. PMID:27145721

  13. The Leaf Epidermome of Catharanthus roseus Reveals Its Biochemical Specialization[W][OA

    PubMed Central

    Murata, Jun; Roepke, Jonathon; Gordon, Heather; De Luca, Vincenzo

    2008-01-01

    Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis–enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed. PMID:18326827

  14. High expression of integrin β6 in association with the Rho-Rac pathway identifies a poor prognostic subgroup within HER2 amplified breast cancers.

    PubMed

    Desai, Krisha; Nair, Madhumathy G; Prabhu, Jyothi S; Vinod, Anupama; Korlimarla, Aruna; Rajarajan, Savitha; Aiyappa, Radhika; Kaluve, Rohini S; Alexander, Annie; Hari, P S; Mukherjee, Geetashree; Kumar, Rekha V; Manjunath, Suraj; Correa, Marjorrie; Srinath, B S; Patil, Shekhar; Prasad, M S N; Gopinath, K S; Rao, Raman N; Violette, Shelia M; Weinreb, Paul H; Sridhar, T S

    2016-08-01

    Integrin αvβ6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin β6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvβ6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin β6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvβ6 integrin by immunohistochemistry (IHC). The expression levels of members of Rho-Rac pathway including downstream genes (ACTR2, ACTR3) and effector proteinases (MMP9, MMP15) were estimated by qPCR in the HER2+ subset (N = 59). There is a significant increase in the mean expression of ITGB6 in HER2+ tumors compared to HR+HER2- and triple negative (TNBC) subtypes (P = 0.00). HER2+ tumors with the highest levels (top quartile) of ITGB6 have significantly elevated levels of all the genes of the Rho-Rac pathway (P-values from 0.01 to 0.0001). Patients in this group have a significantly shorter disease-free survival compared to the group with lower ITGB6 levels (HR = 2.9 (0.9-8.9), P = 0.05). The mean level of ITGB6 expression is increased further in lymph node-positive tumors. The increased regional and distant metastasis observed in HER2+ tumors with high levels of ITGB6 might be mediated by the canonical Rho-Rac pathway through increased expression of MMP9 and MMP15. PMID:27184932

  15. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  16. Pathways for Modulating Exosome Lipids Identified By High-Density Lipoprotein-Like Nanoparticle Binding to Scavenger Receptor Type B-1.

    PubMed

    Angeloni, Nicholas L; McMahon, Kaylin M; Swaminathan, Suchitra; Plebanek, Michael P; Osman, Iman; Volpert, Olga V; Thaxton, C Shad

    2016-01-01

    Exosomes are produced by cells to mediate intercellular communication, and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media, and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1, and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection, and isolation. PMID:26964503

  17. Pathways for Modulating Exosome Lipids Identified By High-Density Lipoprotein-Like Nanoparticle Binding to Scavenger Receptor Type B-1

    PubMed Central

    Angeloni, Nicholas L.; McMahon, Kaylin M.; Swaminathan, Suchitra; Plebanek, Michael P.; Osman, Iman; Volpert, Olga V.; Thaxton, C. Shad

    2016-01-01

    Exosomes are produced by cells to mediate intercellular communication, and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media, and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1, and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection, and isolation. PMID:26964503

  18. Identifying beach sand sources and pathways in the San Francisco Bay Coastal System through the integration of bed characteristics, geochemical tracers, current measurements, and numerical modeling

    NASA Astrophysics Data System (ADS)

    Barnard, P.; Foxgrover, A. C.; Elias, E.; Erikson, L. H.; Hein, J. R.; McGann, M. L.; Mizell, K.; Rosenbauer, R. J.; Swarzenski, P. W.; Takesue, R. K.; Wong, F. L.; Woodrow, D. L.

    2012-12-01

    A unique, multi-faceted provenance study was performed to definitively establish the primary sources, sinks, and transport pathways of beach sized-sand in the San Francisco Bay Coastal System. This integrative program is based on comprehensive surficial sediment sampling of the San Francisco Bay Coastal System, including the seabed, bay floor, area beaches, adjacent rock units, and major drainages. Analyses of sample morphometrics and biological composition (e.g., foraminifera) were then integrated with a suite of tracers including 87Sr/86Sr and 143Nd/144Nd isotopes, rare earth elements, semi-quantitative X-ray diffraction mineralogy, and heavy minerals, and with process-based numerical modeling, in situ current measurements, and bedform asymmetry, to robustly determine the provenance of beach-sized sand in the region. Cross-validating geochemical analyses, numerical modeling, physical process measurements, and proxy-based techniques (e.g., bedform asymmetry, grain size morphometrics) is proved an effective technique for confidently defining sources, pathways, and sinks of sand in complex coastal-estuarine systems. The consensus results highlight the regional impact of a sharp reduction in the primary sediment source, the Sierras, to the San Francisco Bay Coastal System over the last century in driving erosion of the bay floor, ebb-tidal delta, and the outer coast south of the Golden Gate.A) Calculated transport directions based on the integration of the provenance techniques. B) Number of techniques applied for each grid cell to determine the final transport directions.

  19. Probabilistic inference of biochemical reactions in microbial communities from metagenomic sequences.

    PubMed

    Jiao, Dazhi; Ye, Yuzhen; Tang, Haixu

    2013-01-01

    Shotgun metagenomics has been applied to the studies of the functionality of various microbial communities. As a critical analysis step in these studies, biological pathways are reconstructed based on the genes predicted from metagenomic shotgun sequences. Pathway reconstruction provides insights into the functionality of a microbial community and can be used for comparing multiple microbial communities. The utilization of pathway reconstruction, however, can be jeopardized because of imperfect functional annotation of genes, and ambiguity in the assignment of predicted enzymes to biochemical reactions (e.g., some enzymes are involved in multiple biochemical reactions). Considering that metabolic functions in a microbial community are carried out by many enzymes in a collaborative manner, we present a probabilistic sampling approach to profiling functional content in a metagenomic dataset, by sampling functions of catalytically promiscuous enzymes within the context of the entire metabolic network defined by the annotated metagenome. We test our approach on metagenomic datasets from environmental and human-associated microbial communities. The results show that our approach provides a more accurate representation of the metabolic activities encoded in a metagenome, and thus improves the comparative analysis of multiple microbial communities. In addition, our approach reports likelihood scores of putative reactions, which can be used to identify important reactions and metabolic pathways that reflect the environmental adaptation of the microbial communities. Source code for sampling metabolic networks is available online at http://omics.informatics.indiana.edu/mg/MetaNetSam/. PMID:23555216

  20. Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Lescuyer, Arlette; Lancelot, Julien; Dissous, Colette

    2014-01-01

    Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae. PMID:25516836

  1. S-system-based analysis of the robust properties common to many biochemical network models.

    PubMed

    Matsuoka, Yu; Jahan, Nusrat; Kurata, Hiroyuki

    2016-05-01

    Robustness is a key feature to characterize the adaptation of organisms to changes in their internal and external environments. A broad range of kinetic or dynamic models of biochemical systems have been developed. Robustness analyses are attractive for exploring some common properties of many biochemical models. To reveal such features, we transform different types of mathematical equations into a standard or intelligible formula and use the multiple parameter sensitivity (MPS) to identify some factors critically responsible for the total robustness to many perturbations. The MPS would be determined by the top quarter of the highly sensitive parameters rather than the single parameter with the maximum sensitivity. The MPS did not show any correlation to the network size. The MPS is closely related to the standard deviation of the sensitivity profile. A decrease in the standard deviation enhanced the total robustness, which shows the hallmark of distributed robustness that many factors (pathways) involve the total robustness. PMID:26861555

  2. Mechanism of Salinity Tolerance in Plants: Physiological, Biochemical, and Molecular Characterization

    PubMed Central

    Huang, Bingru

    2014-01-01

    Salinity is a major abiotic stress limiting growth and productivity of plants in many areas of the world due to increasing use of poor quality of water for irrigation and soil salinization. Plant adaptation or tolerance to salinity stress involves complex physiological traits, metabolic pathways, and molecular or gene networks. A comprehensive understanding on how plants respond to salinity stress at different levels and an integrated approach of combining molecular tools with physiological and biochemical techniques are imperative for the development of salt-tolerant varieties of plants in salt-affected areas. Recent research has identified various adaptive responses to salinity stress at molecular, cellular, metabolic, and physiological levels, although mechanisms underlying salinity tolerance are far from being completely understood. This paper provides a comprehensive review of major research advances on biochemical, physiological, and molecular mechanisms regulating plant adaptation and tolerance to salinity stress. PMID:24804192

  3. BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS

    EPA Science Inventory

    Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis ...

  4. Biochemical adaptation to ocean acidification.

    PubMed

    Stillman, Jonathon H; Paganini, Adam W

    2015-06-01

    The change in oceanic carbonate chemistry due to increased atmospheric PCO2  has caused pH to decline in marine surface waters, a phenomenon known as ocean acidification (OA). The effects of OA on organisms have been shown to be widespread among diverse taxa from a wide range of habitats. The majority of studies of organismal response to OA are in short-term exposures to future levels of PCO2 . From such studies, much information has been gathered on plastic responses organisms may make in the future that are beneficial or harmful to fitness. Relatively few studies have examined whether organisms can adapt to negative-fitness consequences of plastic responses to OA. We outline major approaches that have been used to study the adaptive potential for organisms to OA, which include comparative studies and experimental evolution. Organisms that inhabit a range of pH environments (e.g. pH gradients at volcanic CO2 seeps or in upwelling zones) have great potential for studies that identify adaptive shifts that have occurred through evolution. Comparative studies have advanced our understanding of adaptation to OA by linking whole-organism responses with cellular mechanisms. Such optimization of function provides a link between genetic variation and adaptive evolution in tuning optimal function of rate-limiting cellular processes in different pH conditions. For example, in experimental evolution studies of organisms with short generation times (e.g. phytoplankton), hundreds of generations of growth under future conditions has resulted in fixed differences in gene expression related to acid-base regulation. However, biochemical mechanisms for adaptive responses to OA have yet to be fully characterized, and are likely to be more complex than simply changes in gene expression or protein modification. Finally, we present a hypothesis regarding an unexplored area for biochemical adaptation to ocean acidification. In this hypothesis, proteins and membranes exposed to the

  5. Biosynthetic pathways of ergot alkaloids.

    PubMed

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  6. Biosynthetic Pathways of Ergot Alkaloids

    PubMed Central

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  7. LC-QTOF-MS-based targeted metabolomics of arginine-creatine metabolic pathway-related compounds in plasma: application to identify potential biomarkers in pediatric chronic kidney disease.

    PubMed

    Benito, Sandra; Sánchez, Alicia; Unceta, Nora; Andrade, Fernando; Aldámiz-Echevarria, Luis; Goicolea, M Aránzazu; Barrio, Ramón J

    2016-01-01

    Chronic kidney disease (CKD) is a major epidemiologic problem which causes several disturbances in adults and in pediatrics. Despite being a worldwide public health problem, information available for CKD in the pediatric population is scarce. For that reason, an ion-pair reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method has been developed and validated in order to analyze 16 amino acids, amino acid derivatives, and analogous compounds related to the arginine-creatine metabolic pathway that are suspicious of being increased or decreased in plasma from patients with CKD. The analytical method involved the addition of dithiothreitol, a reducing agent which reduces disulfide and thus giving total aminothiol concentration, as well as a simple precipitation of plasma proteins. Moreover, despite amino acids being usually derivatized to improve their retention time and to enhance their signal, for this method, an ion-pairing reagent was used, thus avoiding the need for derivatization. Subsequently, analysis of plasma from pediatric patients suffering from CKD and control pediatrics was carried out. As a result, glycine, citrulline, creatinine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) were significantly increased in patients with CKD, regardless of their creatinine level, whereas in addition to these compounds dimethylglycine was also increased when CKD patients had plasma creatinine concentrations above 12 μg mL(-1), thus all are suggested as potential biomarkers for renal impairment. PMID:26537926

  8. Combined Transcriptomic and Proteomic Approach to Identify Toxicity Pathways in Early Life Stages of Japanese Medaka (Oryzias latipes) Exposed to 1,2,5,6-Tetrabromocyclooctane (TBCO).

    PubMed

    Sun, Jianxian; Tang, Song; Peng, Hui; Saunders, David M V; Doering, Jon A; Hecker, Markus; Jones, Paul D; Giesy, John P; Wiseman, Steve

    2016-07-19

    Currently, the novel brominated flame retardant 1,2,5,6-tetrabromocyclooctane (TBCO) is considered a potential replacement for hexabromocyclododecane (HBCD). Therefore, use of TBCO could increase in the near future. To assess potential toxicological risks to aquatic organisms, embryos of Japanese medaka (Oryzias latipes) were exposed to 10, 100, or 1000 μg/L TBCO from 2 h postfertilization until 1 day post-hatch. TBCO accumulated in embryos in the order of 0.43-1.3 × 10(4)-fold, and the rate constant of accumulation was 1.7-1.8 per day. The number of days to hatch and the hatching success of embryos exposed to the medium and the greatest concentrations of TBCO were impaired. Responses of the transcriptome (RNA-seq) and proteome were characterized in embryos exposed to 100 μg/L TBCO because this was the least concentration of TBCO that caused an effect on hatching. Consistent with effects on hatching, proteins whose abundances were reduced by exposure to TBCO were enriched in embryo development and hatching pathways. Also, on the basis of the responses of transcriptome and proteome, it was predicted that TBCO might impair vision and contraction of cardiac muscle, respectively, and these effects were confirmed by targeted bioassays. This study provided a comprehensive understanding of effects of TBCO on medaka at early life stages and illustrated the power of "omics" to explain and predict phenotypic responses to chemicals. PMID:27322799

  9. Heterologous protein production using the twin arginine translocation pathway

    DOEpatents

    Pohlschroder, Mechtild; Kissinger, Jessica C; Rose, R. Wesley; Brueser, Thomas; Dilks, Kieran

    2008-11-04

    Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

  10. Lower glycolysis carries a higher flux than any biochemically possible alternative

    PubMed Central

    Court, Steven J.; Waclaw, Bartlomiej; Allen, Rosalind J.

    2015-01-01

    The universality of many pathways of core metabolism suggests a strong role for evolutionary selection, but it remains unclear whether existing pathways have been selected from a large or small set of biochemical possibilities. To address this question, we construct in silico all possible biochemically feasible alternatives to the trunk pathway of glycolysis and gluconeogenesis, one of the most highly conserved pathways in metabolism. We show that, even though a large number of alternative pathways exist, the alternatives carry lower flux than the real pathway under typical physiological conditions. We also find that if physiological conditions were different, different pathways could outperform those found in nature. Together, our results demonstrate how thermodynamic and biophysical constraints restrict the biochemical alternatives that are open to evolution, and suggest that the existing trunk pathway of glycolysis and gluconeogenesis may represent a maximal flux solution. PMID:26416228

  11. Lower glycolysis carries a higher flux than any biochemically possible alternative.

    PubMed

    Court, Steven J; Waclaw, Bartlomiej; Allen, Rosalind J

    2015-01-01

    The universality of many pathways of core metabolism suggests a strong role for evolutionary selection, but it remains unclear whether existing pathways have been selected from a large or small set of biochemical possibilities. To address this question, we construct in silico all possible biochemically feasible alternatives to the trunk pathway of glycolysis and gluconeogenesis, one of the most highly conserved pathways in metabolism. We show that, even though a large number of alternative pathways exist, the alternatives carry lower flux than the real pathway under typical physiological conditions. We also find that if physiological conditions were different, different pathways could outperform those found in nature. Together, our results demonstrate how thermodynamic and biophysical constraints restrict the biochemical alternatives that are open to evolution, and suggest that the existing trunk pathway of glycolysis and gluconeogenesis may represent a maximal flux solution. PMID:26416228

  12. Nanoparticles as biochemical sensors

    PubMed Central

    El-Ansary, Afaf; Faddah, Layla M

    2010-01-01

    There is little doubt that nanoparticles offer real and new opportunities in many fields, such as biomedicine and materials science. Such particles are small enough to enter almost all areas of the body, including cells and organelles, potentially leading to new approaches in nanomedicine. Sensors for small molecules of biochemical interest are of critical importance. This review is an attempt to trace the use of nanomaterials in biochemical sensor design. The possibility of using nanoparticles functionalized with antibodies as markers for proteins will be elucidated. Moreover, capabilities and applications for nanoparticles based on gold, silver, magnetic, and semiconductor materials (quantum dots), used in optical (absorbance, luminescence, surface enhanced Raman spectroscopy, surface plasmon resonance), electrochemical, and mass-sensitive sensors will be highlighted. The