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Sample records for active gpr17 splice

  1. GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Holst, Birgitte; Petersen, Pia S

    2007-01-01

    nervous system as characterized with both quantitative RT-PCR and in situ hybridization analysis. A functional analysis of the GPR39 promoter region identified sites for the hepatocyte nuclear factors 1alpha and 4alpha (HNF-1alpha and -4alpha) and specificity protein 1 (SP1) transcription factors as being......G protein-coupled receptor 39 (GPR39) is a constitutively active, orphan member of the ghrelin receptor family that is activated by zinc ions. GPR39 is here described to be expressed in a full-length, biologically active seven-transmembrane form, GPR39-1a, as well as in a truncated splice variant...... five-transmembrane form, GPR39-1b. The 3' exon of the GPR39 gene overlaps with an antisense gene called LYPD1 (Ly-6/PLAUR domain containing 1). Quantitative RT-PCR analysis demonstrated that GPR39-1a is expressed selectively throughout the gastrointestinal tract, including the liver and pancreas...

  2. G-Protein-Coupled Receptor Gpr17 Expression in Two Multiple Sclerosis Remyelination Models.

    Science.gov (United States)

    Nyamoya, Stella; Leopold, Patrizia; Becker, Birte; Beyer, Cordian; Hustadt, Fabian; Schmitz, Christoph; Michel, Anne; Kipp, Markus

    2018-06-05

    In multiple sclerosis patients, demyelination is prominent in both the white and gray matter. Chronic clinical deficits are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. The underlying molecular mechanisms of remyelination and its failure remain currently unclear. Recent studies have recognized G protein-coupled receptor 17 (GPR17) as an important regulator of oligodendrocyte development and remyelination. So far, the relevance of GPR17 for myelin repair was mainly tested in remyelinating white matter lesions. The relevance of GPR17 for gray matter remyelination as well as remyelination of chronic white matter lesions was not addressed so far. Here, we provide a detailed characterization of GPR17 expression during experimental de- and remyelination. Experimental lesions with robust and limited endogenous remyelination capacity were established by either acute or chronic cuprizone-induced demyelination. Furthermore, remyelinating lesions were induced by the focal injection of lysophosphatidylcholine (LPC) into the corpus callosum. GPR17 expression was analyzed by complementary techniques including immunohistochemistry, in situ hybridization, and real-time PCR. In control animals, GPR17 + cells were evenly distributed in the corpus callosum and cortex and displayed a highly ramified morphology. Virtually all GPR17 + cells also expressed the oligodendrocyte-specific transcription factor OLIG2. After acute cuprizone-induced demyelination, robust endogenous remyelination was evident in the white matter corpus callosum but not in the gray matter cortex. Endogenous callosal remyelination was paralleled by a robust induction of GPR17 expression which was absent in the gray matter cortex. Higher numbers of GPR17 + cells were as well observed after LPC-induced focal white matter demyelination. In contrast, densities of GPR17 + cells were comparable to control animals after chronic cuprizone-induced demyelination indicating

  3. PI3K/Akt Activated by GPR30 and Src Regulates 17β-Estradiol-Induced Cultured Immature Boar Sertoli Cells Proliferation.

    Science.gov (United States)

    Yang, Wei-Rong; Zhu, Feng-Wei; Zhang, Jiao-Jiao; Wang, Yi; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

    2016-05-24

    Sertoli cell (SC) is a key element in the process of spermatogenesis. Accumulating research show that estrogen plays an important role in regulating boar SC proliferation. However, it is unclear whether phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) is involved in this process. In the present study, the role of PI3K/Akt on the 17β-estradiol-induced piglet SC proliferation was explored. In addition, we also explained the roles of G-protein-coupled estrogen receptor (GPR30) and Sarcoma protein (Src) in this process. Our study demonstrated that, 17β-estradiol induced activation of PI3K in a time-dependent manner. Both G-15 (an antagonist of GPR30, 0.1 μmol/L) and PP2 (an inhibitor of Src, 2.0 μmol/L) inhibited 17β-estradiol-induced activation of PI3K, reduced SC proliferation, and decreased messenger RNA (mRNA) and protein expression of S-phase kinase-associated protein 2 (Skp2). We also found that 17β-estradiol induced activation of Akt in a time-dependent manner. Both LY294002 (an inhibitor of PI3K) and 10-DEBC (an inhibitor of Akt) significantly reduced 17β-estradiol-induced SC proliferation and reduced mRNA and protein expression of Skp2. In addition, LY294002 inhibited 17β-estradiol-induced activation of Akt. The results indicated that 17β-estradiol regulates SC proliferation by activating PI3K/Akt. Both GPR30 and Src are involved in 17β-estradiol-induced phosphorylation of PI3K/Akt. Activation of PI3K/Akt enhances the expression of Skp2, which promotes SC proliferation. © The Author(s) 2016.

  4. Two alternatively spliced GPR39 transcripts in seabream: molecular cloning, genomic organization, and regulation of gene expression by metabolic signals.

    Science.gov (United States)

    Zhang, Yong; Liu, Yun; Huang, Xigui; Liu, Xiaochun; Jiao, Baowei; Meng, Zining; Zhu, Pei; Li, Shuisheng; Lin, Haoran; Cheng, Christopher H K

    2008-12-01

    Two GPR39 transcripts, designated as sbGPR39-1a and sbGPR39-1b, were identified in black seabream (Acanthopagrus schlegeli). The deduced amino acid (aa) sequence of sbGPR39-1a contains 423 residues with seven putative transmembrane (TM) domains. On the other hand, sbGPR39-1b contains 284 aa residues with only five putative TM domains. Northern blot analysis confirmed the presence of two GPR39 transcripts in the seabream intestine, stomach, and liver. Apart from seabream, the presence of two GPR39 transcripts was also found to exist in a number of teleosts (zebrafish and pufferfish) and mammals (human and mouse). Analysis of the GPR39 gene structure in different species suggests that the two GPR39 transcripts are generated by alternative splicing. When the seabream receptors were expressed in cultured HEK293 cells, Zn(2)(+) could trigger sbGPR39-1a signaling through the serum response element pathway, but no such functionality could be detected for the sbGPR39-1b receptor. The two receptors were found to be differentially expressed in seabream tissues. sbGPR39-1a is predominantly expressed in the gastrointestinal tract. On the other hand, sbGPR39-1b is widely expressed in most central and peripheral tissues except muscle and ovary. The expression of sbGPR39-1a in the intestine and the expression of sbGPR39-1b in the hypothalamus were decreased significantly during food deprivation in seabream. On the contrary, the expression of the GH secretagogue receptors (sbGHSR-1a and sbGHSR-1b) was significantly increased in the hypothalamus of the food-deprived seabream. The reciprocal regulatory patterns of expression of these two genes suggest that both of them are involved in controlling the physiological response of the organism during starvation.

  5. The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

    Science.gov (United States)

    Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

    2016-01-08

    Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. EBI2, GPR18 and GPR17--three structurally related, but biologically distinct 7TM receptors

    DEFF Research Database (Denmark)

    Nørregaard, Kristine; Benned-Jensen, Tau; Rosenkilde, Mette Marie

    2011-01-01

    7TM receptors constitute one of the largest superfamilies of proteins in the human genome. They are involved in a large number of physiological and pathological processes in the human body and thus represent major and important drug targets for the pharmaceutical industry. Although the majority...... have been deorphanized, many remain orphan, and these orphan receptors constitute a large pool of potential drug targets. This review focuses on one of these orphan targets, the Epstein-Barr Virus-induced receptor 2, EBI2 (or GPR183), together with two structurally related receptors, GPR17 and GPR18...

  7. Differential local tissue permissiveness influences the final fate of GPR17-expressing oligodendrocyte precursors in two distinct models of demyelination.

    Science.gov (United States)

    Coppolino, Giusy T; Marangon, Davide; Negri, Camilla; Menichetti, Gianluca; Fumagalli, Marta; Gelosa, Paolo; Dimou, Leda; Furlan, Roberto; Lecca, Davide; Abbracchio, Maria P

    2018-05-01

    Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17-iCreER T2 xCAG-eGFP mice) allowing to follow the final fate of GPR17 + cells by tamoxifen-induced GFP-labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP + cells at damaged areas. However, only in the cuprizone model reacting GFP + cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP + cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor-1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti-inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery. © 2018 The Authors GLIA Published by Wiley Periodicals, Inc.

  8. Differential local tissue permissiveness influences the final fate of GPR17‐expressing oligodendrocyte precursors in two distinct models of demyelination

    Science.gov (United States)

    Coppolino, Giusy T.; Marangon, Davide; Negri, Camilla; Menichetti, Gianluca; Fumagalli, Marta; Gelosa, Paolo; Dimou, Leda; Furlan, Roberto; Lecca, Davide

    2018-01-01

    Abstract Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17‐iCreERT2xCAG‐eGFP mice) allowing to follow the final fate of GPR17+ cells by tamoxifen‐induced GFP‐labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP+ cells at damaged areas. However, only in the cuprizone model reacting GFP+ cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP+ cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor‐1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti‐inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery. PMID:29424466

  9. The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair.

    Directory of Open Access Journals (Sweden)

    Davide Lecca

    promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a "sensor" that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.

  10. GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

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    Ranghino Graziella

    2008-06-01

    Full Text Available Abstract Background GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor were then modeled on the receptor. Results Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255. The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. Conclusion Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist

  11. Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells.

    Science.gov (United States)

    Dong, Lixue; Krewson, Elizabeth A; Yang, Li V

    2017-01-27

    Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

  12. A novel splice variant of the stem cell marker LGR5/GPR49 is correlated with the risk of tumor-related death in soft-tissue sarcoma patients

    International Nuclear Information System (INIS)

    Rot, Swetlana; Taubert, Helge; Bache, Matthias; Greither, Thomas; Würl, Peter; Eckert, Alexander W; Schubert, Johannes; Vordermark, Dirk; Kappler, Matthias

    2011-01-01

    The human leucine-rich, repeat-containing G protein-coupled receptor (LGR) 5, also called GPR49, is a marker of stem cells in adult intestinal epithelium, stomach and hair follicles. LGR5/GPR49 is overexpressed in tumors of the colon, ovary and liver and in basal cell carcinomas. Moreover, an expression in skeletal muscle tissues was also detected. However, there has been no investigation regarding the expression and function of LGR5/GPR49 in soft-tissue sarcomas (STS) yet. Seventy-seven frozen tumor samples from adult STS patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of wild type LGR5/GPR49 and a newly identified splice variant of LGR5/GPR49 lacking exon 5 (that we called GPR49Δ5) were quantified. A low mRNA expression level of GPR49Δ5, but not wild type LGR5/GPR49, was significantly correlated with a poor prognosis for the disease-associated survival of STS patients (RR = 2.6; P = 0.026; multivariate Cox's regression hazard analysis). Furthermore, a low mRNA expression level of GPR49Δ5 was associated with a shorter recurrence-free survival (P = 0.043). However, tumor onset in patients with a lower expression level of GPR49Δ5 mRNA occurred 7.5 years later (P = 0.04) than in patients with a higher tumor level of GPR49Δ5 mRNA. An attenuated mRNA level of the newly identified transcript variant GPR49Δ5 is a negative prognostic marker for disease-associated and recurrence-free survival in STS patients. Additionally, a lower GPR49Δ5 mRNA level is associated with a later age of tumor onset. A putative role of GPR49Δ5 expression in tumorigenesis and tumor progression of soft tissue sarcomas is suggested

  13. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells

    KAUST Repository

    Ren, Jian

    2012-03-06

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E 2) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E 2 elevated [Ca 2+] i and increased Ca 2+ oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E 2 mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E 2 activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E 2 induces the non-genomic responses Ca 2+ release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E 2 responses. © 2012 Springer Science+Business Media, LLC.

  14. Forced unbinding of GPR17 ligands from wild type and R255I mutant receptor models through a computational approach

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    Fantucci Piercarlo

    2010-03-01

    Full Text Available Abstract Background GPR17 is a hybrid G-protein-coupled receptor (GPCR activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs, and involved in brain damage and repair. Its exploitment as a target for novel neuro-reparative strategies depends on the elucidation of the molecular determinants driving binding of purinergic and leukotrienic ligands. Here, we applied docking and molecular dynamics simulations (MD to analyse the binding and the forced unbinding of two GPR17 ligands (the endogenous purinergic agonist UDP and the leukotriene receptor antagonist pranlukast from both the wild-type (WT receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I to Ile. Results MD suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and extracellular loop (EL 2. The driving interaction involves R255 and the UDP phosphate moiety. To support this hypothesis, steered MD experiments showed that the energy required to unbind UDP is higher for the WT receptor than for R255I. Three potential binding sites for pranlukast where instead found and analysed. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the WT receptor and the R255I receptor were found for the unbinding of pranlukast. Conclusions These data thus suggest that, in contrast to which has been hypothesized for nucleotides, the lack of the R255 residue doesn't affect the binding of pranlukast a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17.

  15. Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

    Science.gov (United States)

    2014-01-01

    The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [3H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities. PMID:24900835

  16. Development of [(3)H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([(3)H]PSB-12150): A Useful Tool for Studying GPR17.

    Science.gov (United States)

    Köse, Meryem; Ritter, Kirsten; Thiemke, Katharina; Gillard, Michel; Kostenis, Evi; Müller, Christa E

    2014-04-10

    The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.

  17. Orphan receptor GPR110, an oncogene overexpressed in lung and prostate cancer

    International Nuclear Information System (INIS)

    Lum, Amy M; Wang, Bruce B; Beck-Engeser, Gabriele B; Li, Lauri; Channa, Namitha; Wabl, Matthias

    2010-01-01

    GPR110 is an orphan G protein-coupled receptor--a receptor without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. In a retroviral insertion mutagenesis screen in the mouse, we identified GPR110 as an oncogene. This prompted us to study the potential isoforms that can be gleaned from known GPR110 transcripts, and the expression of these isoforms in normal and transformed human tissues. Various epitope-tagged isoforms of GPR110 were expressed in cell lines and assayed by western blotting to determine cleavage, surface localization, and secretion patterns. GPR110 transcript and protein levels were measured in lung and prostate cancer cell lines and clinical samples, respectively, by quantitative PCR and immunohistochemistry. We found four potential splice variants of GPR110. Of these variants, we confirmed three as being expressed as proteins on the cell surface. Isoform 1 is the canonical form, with a molecular mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it had higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is greatly overexpressed in human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential

  18. GPR Use and Activities in Denmark

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    Ringgaard, Jørgen; Wisén, Roger

    2014-05-01

    Academic work on GPR in Denmark is performed both by the Technical University of Denmark (DTU) and the University of Copenhagen (KU). The work at DTU includes development of antennas and systems, e.g. an airborne ice-sounder GPR system (POLARIS) that today is in frequent use for monitoring of ice thickness in Greenland. DTU often collaborates with ESA (European Space Agency) regarding electromagnetic development projects. At KU there is an ongoing work with GPR applied to water resources. The main objective is to study flux of water and matter across different hydrological domains. There are several recent publications from KU describing research for data analysis and modelling as well as hydro geophysical applications. Also the Geological Survey of Denmark and Greenland (GEUS) performs frequent geological mapping with GPR. There have been mainly two actors on the Danish commercial market for several years: FalkGeo and Ramboll. Falkgeo has been active for many years acquiring data for several different applications such as archeology, utilities and roads. Their equipment pool comprises both a multichannel Terravision system form GSSI and a 2D system from Mala Geoscience with a comprehensive range of antennas. Ramboll has performed GPR surveys for two decades mainly with 2D systems from GSSI. In recent years Ramboll has also obtained a system with RTA antennas from Mala Geoscience and a multichannel system from 3D-Radar. These systems have opened markets both for deeper geological mapping and for shallow mapping. The geological mapping with the Mala system has often been combined with resistivity imaging (CVES) and refraction seismic. The 3D system has been applied in airports and on road for mapping of layer thicknesses, delamination and for control of asphalt works. Other areas comprise bridge deck evaluation and utility mapping. Ramboll also acts as client advisor for BaneDanmark, a state owned company who operates and develops the Danish state railway network

  19. The 20S proteasome splicing activity discovered by SpliceMet.

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    Juliane Liepe

    2010-06-01

    Full Text Available The identification of proteasome-generated spliced peptides (PSP revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS. For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

  20. The Proton-Activated Receptor GPR4 Modulates Glucose Homeostasis by Increasing Insulin Sensitivity

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    Luca Giudici

    2013-11-01

    Full Text Available Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8 and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old, the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues.

  1. Deletion of Gpr55 Results in Subtle Effects on Energy Metabolism, Motor Activity and Thermal Pain Sensation.

    Directory of Open Access Journals (Sweden)

    Mikael Bjursell

    Full Text Available The G-protein coupled receptor 55 (GPR55 is activated by cannabinoids and non-cannabinoid molecules and has been speculated to play a modulatory role in a large variety of physiological and pathological processes, including in metabolically perturbed states. We therefore generated male mice deficient in the gene coding for the cannabinoid/lysophosphatidylinositol (LPI receptor Gpr55 and characterized them under normal dietary conditions as well as during high energy dense diet feeding followed by challenge with the CB1 receptor antagonist/GPR55 agonist rimonabant. Gpr55 deficient male mice (Gpr55 KO were phenotypically indistinguishable from their wild type (WT siblings for the most part. However, Gpr55 KO animals displayed an intriguing nocturnal pattern of motor activity and energy expenditure (EE. During the initial 6 hours of the night, motor activity was significantly elevated without any significant effect observed in EE. Interestingly, during the last 6 hours of the night motor activity was similar but EE was significantly decreased in the Gpr55 KO mice. No significant difference in motor activity was detected during daytime, but EE was lower in the Gpr55 KO compared to WT mice. The aforementioned patterns were not associated with alterations in energy intake, daytime core body temperature, body weight (BW or composition, although a non-significant tendency to increased adiposity was seen in Gpr55 KO compared to WT mice. Detailed analyses of daytime activity in the Open Field paradigm unveiled lower horizontal activity and rearing time for the Gpr55 KO mice. Moreover, the Gpr55 KO mice displayed significantly faster reaction time in the tail flick test, indicative of thermal hyperalgesia. The BW-decreasing effect of rimonabant in mice on long-term cafeteria diet did not differ between Gpr55 KO and WT mice. In conclusion, Gpr55 deficiency is associated with subtle effects on diurnal/nocturnal EE and motor activity behaviours but does not

  2. The orphan G protein-coupled receptor GPR139 is activated by the peptides

    DEFF Research Database (Denmark)

    Jensen, Anne Cathrine Nøhr; Shehata, Mohamed A; Hauser, Alexander S

    2017-01-01

    GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively), as well...

  3. The contribution of GPR98 and DFNB31 genes to a Spanish Usher syndrome type 2 cohort.

    Science.gov (United States)

    García-García, Gema; Besnard, Thomas; Baux, David; Vaché, Christel; Aller, Elena; Malcolm, Sue; Claustres, Mireille; Millan, Jose M; Roux, Anne-Françoise

    2013-01-01

    Usher syndrome type 2 (USH2) is an autosomal recessive disease characterized by moderate to severe hearing loss and retinitis pigmentosa. To date, three disease-causing genes have been identified, USH2A, GPR98, and DFNB31, of which USH2A is clearly the major contributor. The aim of this work was to determine the contribution of GPR98 and DFNB31 genes in a Spanish cohort of USH2A negative patients using exhaustive molecular analysis, including sequencing, dosage, and splicing analysis. Linkage analysis was performed to prioritize the gene to study, followed by sequencing of exons and intron-exon boundaries of the selected gene, GPR98 (90 exons) or DFNB31 (12 exons). Functional splicing analyses and comparative genomic hybridization array to detect large rearrangements were performed when appropriate. We confirmed that mutations in GPR98 contribute a significant but minor role to Usher syndrome type 2. In a group of patients referred for molecular diagnosis, 43 had been found to be positive for USH2A mutations, the remaining 19 without USH2A alterations were screened, and seven different mutations were identified in the GPR98 gene in seven patients (five in the homozygous state), of which six were novel. All detected mutations result in a truncated protein; deleterious missense mutations were not found. No pathological mutations were identified in the DFNB31 gene. In Spain, USH2A and GPR98 are responsible for 95.8% and 5.2% of USH2 mutated cases, respectively. DFNB31 plays a minor role in the Spanish population. There was a group of patients in whom no mutation was found. These findings confirm the importance of including at least GPR98 analysis for comprehensive USH2 molecular diagnosis.

  4. Deep intronic GPR143 mutation in a Japanese family with ocular albinism.

    Science.gov (United States)

    Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

    2015-06-10

    Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease.

  5. GPR143 mutations in Chinese patients with ocular albinism type 1.

    Science.gov (United States)

    Jia, Xiuhua; Yuan, Jin; Jia, Xiaoyun; Ling, Shiqi; Li, Shiqiang; Guo, Xiangming

    2017-05-01

    The aim of the present study was to evaluate mutations of the G protein-coupled receptor 143 (GPR143) gene for ocular albinism type 1 (OA1) in Chinese patients. For the current study, 8 patients with OA1 were selected from the database of ocular genetic diseases. Genomic DNA of OA1 was prepared from venous leukocytes collected from the patients. Cycle sequencing was used to analyze the exons and adjacent introns of GPR143. The variation detected was analyzed by bidirectional DNA sequencing and further evaluated in 96 controls using heteroduplex‑single strand conformational polymorphism analysis. Additionally, slit lamp photography of anterior segment, fundus photography and optical coherence tomography (OCT) were performed to identify the clinical features of OA1. In five patients with OA1, 5 GPR143 gene mutations were identified and four of them there were novel mutations. The screening rate is 62.5%, including c.333G>A (p.W111X), c.353G>A (p.G118E) (known mutation), C.658+2T>G (splice mutation), c.215_216insCGCTGC (p.71‑72insAA) and c.17T>C (p. L6P). These mutations were absent in the 96 normal controls. Only one patient with OA1 in the present study was female. Patients with OA1 often have congenital nystagmus, refractive error, severe decline of visual acuity (from 0.1 to 0.4) and foveal hypoplasia. Different degrees of pigment loss were evident in the patients' iris and retina, whereas macular structure was not identified in the OCT examination. The findings of the present study expanded the gene mutation spectrum of GPR143 and investigated the clinical phenotype of patients with OA1 in the Chinese population. Additional evidence for clinical diagnosis was provided along with differential diagnosis and genetic counseling.

  6. GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice

    OpenAIRE

    Anchan, Divya; Clark, Sara; Pollard, Kevin; Vasudevan, Nandini

    2014-01-01

    The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17beta estradiol and its rapid signaling properties such as activation of the extracellular-regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G-1, in the female rat can improv...

  7. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    Energy Technology Data Exchange (ETDEWEB)

    Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  8. Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

    Science.gov (United States)

    Yang, Deng-Liang; Xu, Jia-Wen; Zhu, Jian-Guo; Zhang, Yi-Lin; Xu, Jian-Bang; Sun, Qing; Cao, Xiao-Nian; Zuo, Wu-Lin; Xu, Ruo-Shui; Huang, Jie-Hong; Jiang, Fu-Neng; Zhuo, Yang-Jia; Xiao, Bai-Quan; Liu, Yun-Zhong; Yuan, Dong-Bo; Sun, Zhao-Lin; He, Hui-Chan; Lun, Zhao-Rong; Zhong, Wei-De; Zhou, Wen-Liang

    2017-06-03

    Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17β-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca 2+ release from the endoplasmic reticulum, increased the mitochondrial Ca 2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca 2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP 3 ) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. The orphan G protein-coupled receptor 25 (GPR25) is activated by Apelin and Apela in non-mammalian vertebrates.

    Science.gov (United States)

    Zhang, Jiannan; Wan, Yiping; Fang, Chao; Chen, Junan; Ouyang, Wangan; Li, Juan; Wang, Yajun

    2018-06-22

    G protein-coupled receptor 25 (GPR25) is an orphan G protein-coupled receptor in vertebrates, that has been implicated to be associated with autoimmune diseases and regulate blood pressure in humans. However, the endogenous ligand of GPR25 remains unknown in vertebrates. Here, we reported that in non-mammalian vertebrates (zebrafish, spotted gars, and pigeons), GPR25 could be activated by Apelin and Apela peptides, which are also the two endogenous ligands of vertebrate Apelin receptor (APLNR). Using the pGL3-CRE-luciferase reporter assay and confocal microscopy, we first demonstrated that like APLNR, zebrafish GPR25 expressing in HEK293 cells could be effectively activated by zebrafish Apelin and Apela peptides, leading to the inhibition of forskolin-stimulated cAMP production and receptor internalization. Like zebrafish GPR25, pigeon and spotted gar GPR25 could also be activated by Apelin and Apela, and their activation could inhibit forskolin-induced cAMP accumulation. Interestingly, unlike zebrafish (/spotted gar/pigeon) GPR25, human GPR25 could not be activated by Apelin and Apela under the same experimental conditions. RNA-seq analysis further revealed that GPR25 is expressed in a variety of tissues, including the testes and intestine of zebrafish/spotted gars/humans, implying the potential roles of GPR25 signaling in many physiological processes in vertebrates. Taken together, our data not only provides the first proof that the orphan receptor GPR25 possesses two potential ligands 'Apelin and Apela' and its activation decreases intracellular cAMP levels in non-mammalian vertebrates, but also facilitates to unravel the physiological roles of GPR25 signaling in vertebrates. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Estimating water content in an active landfill with the aid of GPR

    Energy Technology Data Exchange (ETDEWEB)

    Yochim, April, E-mail: ayochim@regionofwaterloo.ca [Region of Waterloo Waste Management Division, 925 Erb Street West, Waterloo, ON N2J 3Z4 (Canada); Zytner, Richard G., E-mail: rzytner@uoguelph.ca [School of Engineering, University of Guelph, Guelph, ON N1G 2W1 (Canada); McBean, Edward A., E-mail: emcbean@uoguelph.ca [School of Engineering, University of Guelph, Guelph, ON N1G 2W1 (Canada); Endres, Anthony L., E-mail: alendres@sciborg.uwaterloo.ca [Dept. of Earth and Environmental Sciences, University of Waterloo, Waterloo, ON N2L 3G1 (Canada)

    2013-10-15

    Highlights: • Limited information in the literature on the use of GPR to measure in situ water content in a landfill. • Developed GPR method allows measurement of in situ water content in a landfill. • Developed GPR method is appealing to waste management professionals operating landfills. - Abstract: Landfill gas (LFG) receives a great deal of attention due to both negative and positive environmental impacts, global warming and a green energy source, respectively. However, predicting the quantity of LFG generated at a given landfill, whether active or closed is difficult due to the heterogeneities present in waste, and the lack of accurate in situ waste parameters like water content. Accordingly, ground penetrating radar (GPR) was evaluated as a tool for estimating in situ water content. Due to the large degree of subsurface heterogeneity and the electrically conductive clay cap covering landfills, both of which affect the transmission of the electromagnetic pulses, there is much scepticism concerning the use of GPR to quantify in situ water content within a municipal landfill. Two landfills were studied. The first landfill was used to develop the measurement protocols, while the second landfill provided a means of confirming these protocols. GPR measurements were initially completed using the surface GPR approach, but the lack of success led to the use of borehole (BH) GPR. Both zero offset profiling (ZOP) and multiple offset gathers (MOG) modes were tried, with the results indicating that BH GPR using the ZOP mode is the most simple and efficient method to measure in situ water content. The best results were obtained at a separation distance of 2 m, where higher the water content, smaller the effective separation distance. However, an increase in water content did appear to increase the accuracy of the GPR measurements. For the effective separation distance of 2 m at both landfills, the difference between GPR and lab measured water contents were reasonable

  11. Estimating water content in an active landfill with the aid of GPR

    International Nuclear Information System (INIS)

    Yochim, April; Zytner, Richard G.; McBean, Edward A.; Endres, Anthony L.

    2013-01-01

    Highlights: • Limited information in the literature on the use of GPR to measure in situ water content in a landfill. • Developed GPR method allows measurement of in situ water content in a landfill. • Developed GPR method is appealing to waste management professionals operating landfills. - Abstract: Landfill gas (LFG) receives a great deal of attention due to both negative and positive environmental impacts, global warming and a green energy source, respectively. However, predicting the quantity of LFG generated at a given landfill, whether active or closed is difficult due to the heterogeneities present in waste, and the lack of accurate in situ waste parameters like water content. Accordingly, ground penetrating radar (GPR) was evaluated as a tool for estimating in situ water content. Due to the large degree of subsurface heterogeneity and the electrically conductive clay cap covering landfills, both of which affect the transmission of the electromagnetic pulses, there is much scepticism concerning the use of GPR to quantify in situ water content within a municipal landfill. Two landfills were studied. The first landfill was used to develop the measurement protocols, while the second landfill provided a means of confirming these protocols. GPR measurements were initially completed using the surface GPR approach, but the lack of success led to the use of borehole (BH) GPR. Both zero offset profiling (ZOP) and multiple offset gathers (MOG) modes were tried, with the results indicating that BH GPR using the ZOP mode is the most simple and efficient method to measure in situ water content. The best results were obtained at a separation distance of 2 m, where higher the water content, smaller the effective separation distance. However, an increase in water content did appear to increase the accuracy of the GPR measurements. For the effective separation distance of 2 m at both landfills, the difference between GPR and lab measured water contents were reasonable

  12. Structural basis for constitutive activity and agonist-induced activation of the enteroendocrine fat sensor GPR119

    DEFF Research Database (Denmark)

    Engelstoft, Maja Storm; Norn, C; Pedersen, Maria Hauge

    2014-01-01

    BACKGROUND AND PURPOSE: GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts...... activation (AR231453 and OEA). Novel Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray structures and fully flexible ligand docking. KEY RESULTS: The increased signalling induced by increasing the cell surface expression of GPR119...... in the absence of agonist and the inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the mutational map for residues...

  13. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

    Science.gov (United States)

    Miyagi, Tatsuhiro; Tanaka, Shigeru; Hide, Izumi; Shirafuji, Toshihiko; Sakai, Norio

    2016-01-01

    G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.

  14. Hypothalamic GPR40 Signaling Activated by Free Long Chain Fatty Acids Suppresses CFA-Induced Inflammatory Chronic Pain

    OpenAIRE

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2013-01-01

    GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently inc...

  15. A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

    Science.gov (United States)

    Gagnon, Lyne; Leduc, Martin; Thibodeau, Jean-Francois; Zhang, Ming-Zhi; Grouix, Brigitte; Sarra-Bournet, Francois; Gagnon, William; Hince, Kathy; Tremblay, Mikaël; Geerts, Lilianne; Kennedy, Christopher R J; Hébert, Richard L; Gutsol, Alex; Holterman, Chet E; Kamto, Eldjonai; Gervais, Liette; Ouboudinar, Jugurtha; Richard, Jonathan; Felton, Alexandra; Laverdure, Alexandre; Simard, Jean-Christophe; Létourneau, Sylvie; Cloutier, Marie-Pier; Leblond, Francois A; Abbott, Shaun D; Penney, Christopher; Duceppe, Jean-Simon; Zacharie, Boulos; Dupuis, Jocelyn; Calderone, Angelino; Nguyen, Quang T; Harris, Raymond C; Laurin, Pierre

    2018-05-01

    Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. IL-1β and TNFα inhibit GPR120 (FFAR4) and stimulate GPR84 (EX33) and GPR41 (FFAR3) fatty acid receptor expression in human adipocytes: implications for the anti-inflammatory action of n-3 fatty acids.

    Science.gov (United States)

    Muredda, Laura; Kępczyńska, Małgorzata A; Zaibi, Mohamed S; Alomar, Suliman Y; Trayhurn, Paul

    2018-05-01

    Regulation of the expression of GPCR fatty acid receptor genes has been examined in human adipocytes differentiated in culture. TNFα and IL-1β induced a marked reduction in GPR120 expression, mRNA level falling 17-fold at 24 h in adipocytes incubated with TNFα. In contrast, GPR84 mRNA was dramatically increased by these cytokines (>500-fold for IL-1β at 4 h); GPR41 expression was also stimulated. Rosiglitazone did not affect GPR84 expression, but GPR120 and GPR41 expression increased. Dexamethasone, insulin, linoleic and docosahexaenoic acids (DHA), and TUG891 (GPR120 agonist) had little effect on GPR120 and GPR84 expression. TUG891 did not attenuate the pro-inflammatory actions of TNFα and IL-1β. DHA slightly countered the actions of IL-1β on CCL2, IL6 and ADIPOQ expression, though not on secretion of these adipokines. GPR120 and GP84 gene expression in human adipocytes is highly sensitive to pro-inflammatory mediators; the inflammation-induced inhibition of GPR120 expression may compromise the anti-inflammatory action of GPR120 agonists.

  17. The acute glucose lowering effect of specific GPR120 activation in mice is mainly driven by glucagon-like peptide 1.

    Directory of Open Access Journals (Sweden)

    Linda Sundström

    Full Text Available The mechanism behind the glucose lowering effect occurring after specific activation of GPR120 is not completely understood. In this study, a potent and selective GPR120 agonist was developed and its pharmacological properties were compared with the previously described GPR120 agonist Metabolex-36. Effects of both compounds on signaling pathways and GLP-1 secretion were investigated in vitro. The acute glucose lowering effect was studied in lean wild-type and GPR120 null mice following oral or intravenous glucose tolerance tests. In vitro, in GPR120 overexpressing cells, both agonists signaled through Gαq, Gαs and the β-arrestin pathway. However, in mouse islets the signaling pathway was different since the agonists reduced cAMP production. The GPR120 agonists stimulated GLP-1 secretion both in vitro in STC-1 cells and in vivo following oral administration. In vivo GPR120 activation induced significant glucose lowering and increased insulin secretion after intravenous glucose administration in lean mice, while the agonists had no effect in GPR120 null mice. Exendin 9-39, a GLP-1 receptor antagonist, abolished the GPR120 induced effects on glucose and insulin following an intravenous glucose challenge. In conclusion, GLP-1 secretion is an important mechanism behind the acute glucose lowering effect following specific GPR120 activation.

  18. Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jun; Byrne, Noel; Wang, John; Bricogne, Gerard; Brown, Frank K.; Chobanian, Harry R.; Colletti, Steven L.; Di Salvo, Jerry; Thomas-Fowlkes, Brande; Guo, Yan; Hall, Dawn L.; Hadix, Jennifer; Hastings, Nicholas B.; Hermes, Jeffrey D.; Ho, Thu; Howard, Andrew D.; Josien, Hubert; Kornienko, Maria; Lumb, Kevin J.; Miller, Michael W.; Patel, Sangita B.; Pio, Barbara; Plummer, Christopher W.; Sherborne, Bradley S.; Sheth, Payal; Souza, Sarah; Tummala, Srivanya; Vonrhein, Clemens; Webb, Maria; Allen, Samantha J.; Johnston, Jennifer M.; Weinglass, Adam B.; Sharma, Sujata; Soisson, Stephen M. (Merck); (Globel Phasing)

    2017-06-05

    Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

  19. Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

    Directory of Open Access Journals (Sweden)

    Kazuo Nakamoto

    Full Text Available GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA. However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP, an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol, a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg and GW9508 (1.0 µg, a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg, a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long

  20. Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

    Science.gov (United States)

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2013-01-01

    GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty

  1. Structure and biological activity of endogenous and synthetic agonists of GPR119

    Science.gov (United States)

    Tyurenkov, I. N.; Ozerov, A. A.; Kurkin, D. V.; Logvinova, E. O.; Bakulin, D. A.; Volotova, E. V.; Borodin, D. D.

    2018-02-01

    A G-protein-coupled receptor, GPR119, is a promising pharmacological target for a new class of hypoglycaemic drugs with an original mechanism of action, namely, increase in the glucose-dependent incretin and insulin secretion. In 2005, the first ligands were found and in the subsequent years, a large number of GPR119 agonists were synthesized in laboratories in various countries; the safest and most promising agonists have entered phase I and II clinical trials as agents for the treatment of type 2 diabetes mellitus and obesity. The review describes the major endogenous GPR119 agonists and the main trends in the design and modification of synthetic structures for increasing the hypoglycaemic activity. The data on synthetic agonists are arranged according to the type of the central core of the molecules. The bibliography includes 104 references.

  2. GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice.

    Science.gov (United States)

    Anchan, Divya; Clark, Sara; Pollard, Kevin; Vasudevan, Nandini

    2014-01-01

    The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17β estradiol and its rapid signaling properties such as activation of the extracellular-regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G-1, in the female rat can improve spatial memory, suggesting that GPR30 plays a role in hippocampal-dependent cognition. In this study, we investigated the effect of a similar chronic administration of G-1 on behaviors that denote anxiety in adult ovariectomized female mice, using the elevated plus maze (EPM) and the open field test as well as the activation of the ERK pathway in the hippocampus. Although estradiol benzoate had no effect on behaviors in the EPM or the open field, G-1 had an anxiolytic effect solely in the open field that was independent of ERK signaling in either the ventral or dorsal hippocampus. Such an anxiolytic effect may underlie the ability of G-1 to increase spatial memory, by acting on the hippocampus.

  3. GPR119 agonists: a promising approach for T2DM treatment? A SWOT analysis of GPR119.

    Science.gov (United States)

    Kang, Sang-Uk

    2013-12-01

    Ever since its advent as a promising therapeutic target for type 2 diabetes mellitus (T2DM), G-protein-coupled receptor 119 (GPR119) has received much interest from the pharmaceutical industry. This interest peaked in June 2010, when Sanofi-Aventis agreed to pay Metabolex (Cymabay Therapeutics) US$375 million for MBX-2982, which was a representative orally active GPR119 agonist. However, Sanofi-Aventis opted to terminate the deal in May 2011 and another leading GPR119 agonist, GSK1292263, had a loss of efficacy during its clinical trial. In this review, I discuss the pros and cons of GPR119 through a strengths, weaknesses, opportunities, and threats (SWOT) analysis and propose development strategies for the eventual success of a GPR119 agonist development program. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Spatiotemporal Control of GPR37 Signaling and Its Behavioral Effects by Optogenetics

    Directory of Open Access Journals (Sweden)

    Wu Zheng

    2018-03-01

    Full Text Available Despite the progress in deorphanization of G Protein-Coupled Receptors (GPCRs, ≈100 GPCRs are still classified as orphan receptors without identified endogenous ligands and with unknown physiological functions. The lack of endogenous ligands triggering GPCR signaling has hampered the study of orphan GPCR functions. Using GPR37 as an example, we provide here the first demonstration of the channelrhodopsin 2 (ChR2-GPCR approach to bypass the endogenous ligand and selectively activate the orphan GPCR signal by optogenetics. Inspired by the opto-XR approach, we designed the ChR2-GPR37 chimera, in which the corresponding parts of GPR37 replaced the intracellular portions of ChR2. We showed that optogenetic activation of ChR2/opto-GPR37 elicited specific GPR37 signaling, as evidenced by reduced cAMP level, enhanced ERK phosphorylation and increased motor activity, confirming the specificity of opto-GPR37 signaling. Besides, optogenetic activation of opto-GPR37 uncovered novel aspects of GPR37 signaling (such as IP-3 signaling and anxiety-related behavior. Optogenetic activation of opto-GPR37 permits the causal analysis of GPR37 activity in the defined cells and behavioral responses of freely moving animals. Importantly, given the evolutionarily conserved seven-helix transmembrane structures of ChR2 and orphan GPCRs, we propose that opto-GPR37 approach can be readily applied to other orphan GPCRs for their deorphanization in freely moving animals.

  5. GPR use and activities in the Czech Republic

    Science.gov (United States)

    Stryk, Josef; Matula, Radek

    2014-05-01

    In the field of civil engineering applications in the Czech Republic, Ground Penetrating Radar (GPR) is used particularly for the diagnostics of roads and bridges. There is no producer of GPR in the Czech Republic, sets of different producers are used, particularly Geophysical Survey Systems, Inc. (USA) and MALÅ GeoScience (Sweden). The measurement results are mostly processed by software Radan, Road Doctor Pro, ReflexW and RadEx. The only technical specification in the Czech Republic is TP 233 issued by the Ministry of Transport, which describes the diagnostics of roads by GPR. Apart from a basic description of the method and a measurement system, it mentions possible applications. The only application where accuracy is mentioned is the locating of dowels and tie bars in concrete road pavements, which states that if calibration is performed, the expected depth accuracy is up to 1.0 cm. The following R&D project is currently in progress: New diagnostics methods as a supporting decision tool for maintenance and repair of road pavements - their contribution and ways of their usage (2012-2014) The project aims to test possible non-destructive methods (particularly GPR and laser scanning), make recommendations when and how to use specific methods for individual applications and for changes in technical specifications. The following R&D projects have been recently completed: Position of dowels and tie bars in rigid pavements and importance of their correct placement to pavement performance and service life (2012-2013) The project included an analysis of individual NDT methods used for the location of dowels and tie bars and for testing of their accuracy - GPR, MIT-scan and GPR in combination with a metal detector. Multichannel ground penetrating radar as a tool for monitoring of road and bridge structures (2009-2011) The project included detection of hollow spaces under non-reinforced concrete pavements, detection of excessive amount of water in road construction

  6. Endothelial GPR124 Exaggerates the Pathogenesis of Atherosclerosis by Activating Inflammation

    Directory of Open Access Journals (Sweden)

    Dong-Mei Gong

    2018-01-01

    Full Text Available Background/Aims: Endothelial cell dysfunction is the principal pathological process underlying atherosclerotic cardiovascular disease. G protein-coupled receptor 124 (GPR124, an orphan receptor in the adhesion GPCR subfamily, promotes angiogenesis in the brain. In the present study, we explored the role of endothelial GPR124 in the development and progression of atherosclerosis in adult mice. Methods: Using tetracycline-inducible transgenic systems, we generated mice expressing GPR124 specifically under control of the Tie-2 promoter. The animal model of atherosclerosis was constructed by intravenously injecting AAV-PCSK9DY into tetracycline-regulated mice and feeding the mice a high-fat diet for 16 consecutive weeks. Biochemical analysis and immunohistochemistry methods were used to address the role and mechanism of GPR124 in the pathological process of atherosclerosis. Results: Higher TC (total cholesterol and LDL-C (low density lipoprotein cholesterol levels in serum and greater lipid deposition in the aortic sinus were found in atherosclerotic mice with GPR124 overexpression, coincident with the elevated proliferation of smooth muscle cells. We observed an elevation of ONOO- in the aortic sinus in this model by using immunofluorescence, and the experiments showed that the specific overexpression of GPR124 in the endothelium induced the up-regulation of CD68, NLRP3 and caspase-1 levels in the aortic sinus. Conclusion: The above results indicate that manipulating GPR124 in the endothelium may contribute to delayed pathological progression of atherosclerosis.

  7. Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons.

    Science.gov (United States)

    Wellhauser, Leigh; Belsham, Denise D

    2014-03-27

    Overnutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood. Here we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis. Importantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor-β-activated

  8. Extracellular pH regulates zinc signaling via an Asp residue of the zinc-sensing receptor (ZnR/GPR39).

    Science.gov (United States)

    Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

    2012-09-28

    Zinc activates a specific Zn(2+)-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca(2+) responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na(+)/H(+) exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn(2+) binding site, His(17) or His(19), or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp(313) with alanine resulted in similar Ca(2+) responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na(+)/H(+) exchange at pH 7.4 and pH 6.5. Substitution of Asp(313) to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp(313), which was shown to modulate Zn(2+) binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.

  9. Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)*

    Science.gov (United States)

    Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

    2012-01-01

    Zinc activates a specific Zn2+-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca2+ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na+/H+ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn2+ binding site, His17 or His19, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp313 with alanine resulted in similar Ca2+ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na+/H+ exchange at pH 7.4 and pH 6.5. Substitution of Asp313 to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp313, which was shown to modulate Zn2+ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity. PMID:22879599

  10. The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue [Department of Obstetrics and Gynecology, The International Peace Maternity and Child Health Hospital of the China Welfare Institute Affiliated to Shanghai Jiao Tong University, 910, Hengshan Road, Shanghai (China); Wan, Xiao-Ping, E-mail: wanxiaoping61@126.com [Department of Obstetrics and Gynecology, The International Peace Maternity and Child Health Hospital of the China Welfare Institute Affiliated to Shanghai Jiao Tong University, 910, Hengshan Road, Shanghai (China); He, Yin-Yan [Department of Obstetrics and Gynecology, Shanghai First People' s Hospital, Shanghai Jiao Tong University, Shanghai (China)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. Black-Right-Pointing-Pointer GPR30 mediates the proliferative effects induced by OHT. Black-Right-Pointing-Pointer GPR30 mediates the invasive effects induced by OHT. Black-Right-Pointing-Pointer GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17{beta}-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.

  11. The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

    International Nuclear Information System (INIS)

    Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue; Wan, Xiao-Ping; He, Yin-Yan

    2012-01-01

    Highlights: ► We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. ► GPR30 mediates the proliferative effects induced by OHT. ► GPR30 mediates the invasive effects induced by OHT. ► GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17β-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.

  12. Regulation of energy homeostasis via GPR120

    Directory of Open Access Journals (Sweden)

    Atsuhiko eIchimura

    2014-07-01

    Full Text Available Free fatty acids (FFAs are fundamental units of key nutrients. FFAs exert various biological functions, depending on the chain length and degree of desaturation. Recent studies have shown that several FFAs act as ligands of G-protein-coupled receptors (GPCRs, activate intracellular signaling and exert physiological functions via these GPCRs. GPR120 (also known as free fatty acid receptor 4, FFAR4 is activated by unsaturated medium- to long-chain FFAs and has a critical role in various physiological homeostasis mechanisms such as incretin hormone secretion, food preference, anti-inflammation and adipogenesis. Recent studies showed that a lipid sensor GPR120 has a key role in sensing dietary fat in white adipose tissue and regulates the whole body energy homeostasis in both humans and rodents. Genetic study in human identified the loss-of-functional mutation of GPR120 associated with obesity and insulin resistance. In addition, dysfunction of GPR120 has been linked as a novel risk factor for diet-induced obesity. This review aims to provide evidence from the recent development in physiological function of GPR120 and discusses its functional roles in regulation of energy homeostasis and its potential as drug targets.

  13. The SCFA receptor GPR43 and energy metabolism

    Directory of Open Access Journals (Sweden)

    Ikuo eKimura

    2014-06-01

    Full Text Available Free fatty acids (FFAs are essential nutrients and act as signaling molecules in various cellular processes via binding with FFA receptors. Of these receptors, GPR43 is activated by short chain fatty acids (SCFAs; e.g., acetate, propionate, and butyrate. During feeding, SCFAs are produced by microbial fermentation of dietary fiber in the gut, and these SCFAs become important energy sources for the host. The gut microbiota affects nutrient acquisition and energy regulation of the host and can influence the development of obesity, insulin resistance, and diabetes. Recently, GPR43 has been reported to regulate host energy homeostasis in the gastrointestinal tract and adipose tissues. Hence, GPR43 is also thought to be a potential drug target for metabolic disorders, such as obesity and diabetes. In this review, we summarize the identification, structure, and activities of GPR43, with a focus on host energy regulation, and present an essential overview of our current understanding of its physiological roles in host energy regulation that is mediated by gut microbiota. We also discuss the potential for GPR43 as a therapeutic target.

  14. The Zinc Sensing Receptor, ZnR/GPR39, in Health and Disease

    Directory of Open Access Journals (Sweden)

    Michal Hershfinkel

    2018-02-01

    Full Text Available A distinct G-protein coupled receptor that senses changes in extracellular Zn2+, ZnR/GPR39, was found in cells from tissues in which Zn2+ plays a physiological role. Most prominently, ZnR/GPR39 activity was described in prostate cancer, skin keratinocytes, and colon epithelial cells, where zinc is essential for cell growth, wound closure, and barrier formation. ZnR/GPR39 activity was also described in neurons that are postsynaptic to vesicular Zn2+ release. Activation of ZnR/GPR39 triggers Gαq-dependent signaling and subsequent cellular pathways associated with cell growth and survival. Furthermore, ZnR/GPR39 was shown to regulate the activity of ion transport mechanisms that are essential for the physiological function of epithelial and neuronal cells. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

  15. Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets

    DEFF Research Database (Denmark)

    Ulven, Trond

    2012-01-01

    The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber...... produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence...... in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets....

  16. PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    Hyo-Sup Kim

    Full Text Available BACKGROUND: It has been reported that peroxisome proliferator-activated receptor (PPAR-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells. METHODS: Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ. RESULTS: PPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION: These observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

  17. Characterising pharmacological ligands to study the long chain fatty acid receptors GPR40/FFA1 and GPR120/FFA4

    DEFF Research Database (Denmark)

    Milligan, G; Alvarez-Curto, E; Watterson, K R

    2015-01-01

    The G protein-coupled receptors FFA1 (previously designated GPR40) and FFA4 (previously GPR120) are both activated by saturated and unsaturated longer-chain free fatty acids. With expression patterns and functions anticipated to directly or indirectly promote insulin secretion, provide homeostati...

  18. GPR81, a Cell-Surface Receptor for Lactate, Regulates Intestinal Homeostasis and Protects Mice from Experimental Colitis.

    Science.gov (United States)

    Ranganathan, Punithavathi; Shanmugam, Arulkumaran; Swafford, Daniel; Suryawanshi, Amol; Bhattacharjee, Pushpak; Hussein, Mohamed S; Koni, Pandelakis A; Prasad, Puttur D; Kurago, Zoya B; Thangaraju, Muthusamy; Ganapathy, Vadivel; Manicassamy, Santhakumar

    2018-03-01

    At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation. Copyright © 2018 by The American Association of Immunologists, Inc.

  19. GPR17调控脑白质损伤后内在修复潜能的研究进展%Role of GPR17 in regulating inherent self-repairing potential after cerebral white matter injury

    Institute of Scientific and Technical Information of China (English)

    毛凤霞

    2012-01-01

    脑白质损伤可导致髓鞘损害和神经纤维不能髓鞘化,是脑瘫、智力和视听障碍的主要原因.目前,促进神经再生的内在修复潜能正趋向于成为治疗脑白质损伤的重要策略.近来发现一种新的P2Y受体--GPR17可能调控脑白质损伤后的内在修复潜能.文章将就脑白质的解剖特点、少突胶质细胞的生物学特性及GPR17在脑白质损伤后内在修复中的作用进行综述.%Apnea of prematurity (AOP) , commonly affecting premature infants, is one of the main clinical problems in neonatal intensive care unit. The mechanism of AOP pathogenesis is not clear, with speculation currently focuses on prematurity of respiratory center in preterm infants. Other important factors include altered ventilatory responses to hypoxia and hypercapnia, overinhibition of pulmonary stretch reflex and hypersensitivity to inhibitory neu-rotransmitters. In addition, neonatal diseases may play an additive role in increasing the incidence of apnea. Standard clinical management of AOP includes prone positioning, continuous positive or nasal intermittent positive pressure ventilation and methylxanthine therapy. Other therapies, including sensory stimulations, red blood cell transfusions, and C02 inhalation require further study. This review focuses on the insights into the pathogenesis basis for AOP, and the mechanisms underlying for its therapies.

  20. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    Science.gov (United States)

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

  1. Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping

    Czech Academy of Sciences Publication Activity Database

    Divina, Petr; Kvitkovicova, Andrea; Buratti, E.; Vorechovsky, I.

    2009-01-01

    Roč. 17, č. 6 (2009), s. 759-765 ISSN 1018-4813 Institutional research plan: CEZ:AV0Z50520514 Keywords : mutation * cryptic splice site * exon skipping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.564, year: 2009

  2. Embelin and its derivatives unravel the signaling, proinflammatory and antiatherogenic properties of GPR84 receptor.

    Science.gov (United States)

    Gaidarov, Ibragim; Anthony, Todd; Gatlin, Joel; Chen, Xiaohua; Mills, David; Solomon, Michelle; Han, Sangdon; Semple, Graeme; Unett, David J

    2018-05-01

    GPR84 is an orphan G-protein coupled receptor, expressed on monocytes, macrophages and neutrophils and is significantly upregulated by inflammatory stimuli. The physiological role of GPR84 remains largely unknown. Medium chain fatty acids (MCFA) activate the receptor and have been proposed to be its endogenous ligands, although the high concentrations of MCFAs required for receptor activation generally exceed normal physiological levels. We identified the natural product embelin as a highly potent and selective surrogate GPR84 agonist (originally disclosed in patent application WO2007027661A2, 2007) and synthesized close structural analogs with widely varying receptor activities. These tools were used to perform a comprehensive study of GPR84 signaling and function in recombinant cells and in primary human macrophages and neutrophils. Activation of recombinant GPR84 by embelin in HEK293 cells results in G i/o as well as G12/13-Rho signaling. In human macrophages, GPR84 initiates PTX sensitive Erk1/2 and Akt phosphorylation, PI-3 kinase activation, calcium flux, and release of prostaglandin E2. In addition, GPR84 signaling in macrophages elicits G i Gβγ-mediated augmentation of intracellular cAMP, rather than the decrease expected from G iα engagement. GPR84 activation drives human neutrophil chemotaxis and primes them for amplification of oxidative burst induced by FMLP and C5A. Loss of GPR84 is associated with attenuated LPS-induced release of proinflammatory mediators IL-6, KC-GROα, VEGF, MIP-2 and NGAL from peritoneal exudates. While initiating numerous proinflammatory activities in macrophages and neutrophils, GPR84 also possesses GPR109A-like antiatherosclerotic properties in macrophages. Macrophage receptor activation leads to upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulates reverse cholesterol transport. These data suggest that GPR84 may be a target of therapeutic value and that distinct modes of receptor modulation (inhibition vs

  3. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  4. Lauric Acid Stimulates Mammary Gland Development of Pubertal Mice through Activation of GPR84 and PI3K/Akt Signaling Pathway.

    Science.gov (United States)

    Meng, Yingying; Zhang, Jing; Zhang, Fenglin; Ai, Wei; Zhu, Xiaotong; Shu, Gang; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Liang, Xingwei; Jiang, Qingyan; Wang, Songbo

    2017-01-11

    It has been demonstrated that dietary fat affects pubertal mammary gland development. However, the role of lauric acid (LA) in this process remains unclear. Thus, this study aimed to investigate the effects of LA on mammary gland development in pubertal mice and to explore the underlying mechanism. In vitro, 100 μM LA significantly promoted proliferation of mouse mammary epithelial cell line HC11 by regulating expression of proliferative markers (cyclin D1/3, p21, PCNA). Meanwhile, LA activated the G protein-coupled receptor 84 (GPR84) and PI3K/Akt signaling pathway. In agreement, dietary 1% LA enhanced mammary duct development, increased the expression of GPR84 and cyclin D1, and activated PI3K/Akt in mammary gland of pubertal mice. Furthermore, knockdown of GPR84 or inhibition of PI3K/Akt totally abolished the promotion of HC11 proliferation induced by LA. These results showed that LA stimulated mammary gland development of pubertal mice through activation of GPR84 and PI3K/Akt signaling pathway.

  5. Extracting and identifying concrete structural defects in GPR images

    Science.gov (United States)

    Ye, Qiling; Jiao, Liangbao; Liu, Chuanxin; Cao, Xuehong; Huston, Dryver; Xia, Tian

    2018-03-01

    Traditionally most GPR data interpretations are performed manually. With the advancement of computing technologies, how to automate GPR data interpretation to achieve high efficiency and accuracy has become an active research subject. In this paper, analytical characterizations of major defects in concrete structures, including delamination, air void and moisture in GPR images, are performed. In the study, the image features of different defects are compared. Algorithms are developed for defect feature extraction and identification. For validations, both simulation results and field test data are utilized.

  6. The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

    Science.gov (United States)

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

    2012-01-01

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

  7. Discovery of a potent and selective GPR120 agonist

    DEFF Research Database (Denmark)

    Shimpukade, Bharat; Hudson, Brian D; Hovgaard, Christine Kiel

    2012-01-01

    GPR120 is a receptor of unsaturated long-chain fatty acids reported to mediate GLP-1 secretion, insulin sensitization, anti-inflammatory, and anti-obesity effects and is therefore emerging as a new potential target for treatment of type 2 diabetes and metabolic diseases. Further investigation...... is however hindered by the lack of suitable receptor modulators. Screening of FFA1 ligands provided a lead with moderate activity on GPR120 and moderate selectivity over FFA1. Optimization led to the discovery of the first potent and selective GPR120 agonist....

  8. A novel G protein-coupled receptor of Schistosoma mansoni (SmGPR-3 is activated by dopamine and is widely expressed in the nervous system.

    Directory of Open Access Journals (Sweden)

    Fouad El-Shehabi

    Full Text Available Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3 that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of "orphan" amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs.

  9. GPR Technologies and Methodologies in Italy: A Review

    Science.gov (United States)

    Benedetto, Andrea; Frezza, Fabrizio; Manacorda, Guido; Massa, Andrea; Pajewski, Lara

    2014-05-01

    GPR techniques and technologies have been subject of intense research activities at the Italian level in the last 15 years because of their potential applications specifically to civil engineering. More in detail, several innovative approaches and models have been developed to inspect road pavements to measure the thickness of their layers as well as to diagnose or prevent damage. Moreover, new frontiers in bridge inspection as well as in geotechnical applications such as slides and flows have been investigated using GPR. From the methodological viewpoint, innovative techniques have been developed to solve GPR forward-scattering problems, as well to locate and classify subsurface targets in real-time and to retrieve their properties through multi-resolution strategies, and linear and non-linear methodologies. Furthermore, the application of GPR and other non-destructive testing methods in archaeological prospecting, cultural heritage diagnostics, and in the localization and detection of vital signs of trapped people has been widely investigated. More recently, new theoretical and empirical paradigms regarding water moisture evaluation in various porous media and soil characterization have been published as the results of long terms research activities. Pioneer studies are also currently under development with the scope to correlate GPR measurement with mechanical characteristics of bound and unbound construction materials. In such a framework, this abstract will be aimed at reviewing some of the most recent advances of GPR techniques and technologies within the Italian industrial and academic communities [also including their application within international projects such as FP7 ISTIMES (http://www.istimes.eu)], and at envisaging some of the most promising research trends currently under development. Acknowledgment - This work was supported by COST Action TU1208 'Civil Engineering Applications of Ground Penetrating Radar' References [1] M. Balsi, S. Esposito, F

  10. Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

    Directory of Open Access Journals (Sweden)

    Janna eBlechman

    2013-05-01

    Full Text Available Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

  11. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

    Directory of Open Access Journals (Sweden)

    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  12. Regulating the effects of GPR21, a novel target for type 2 diabetes

    Science.gov (United States)

    Leonard, Siobhán; Kinsella, Gemma K.; Benetti, Elisa; Findlay, John B. C.

    2016-05-01

    Type 2 diabetes is a chronic metabolic disorder primarily caused by insulin resistance to which obesity is a major contributor. Expression levels of an orphan G protein-coupled receptor (GPCR), GPR21, demonstrated a trend towards a significant increase in the epididymal fat pads of wild type high fat high sugar (HFHS)-fed mice. To gain further insight into the potential role this novel target may play in the development of obesity-associated type 2 diabetes, the signalling capabilities of the receptor were investigated. Overexpression studies in HEK293T cells revealed GPR21 to be a constitutively active receptor, which couples to Gαq type G proteins leading to the activation of mitogen activated protein kinases (MAPKs). Overexpression of GPR21 in vitro also markedly attenuated insulin signalling. Interestingly, the effect of GPR21 on the MAPKs and insulin signalling was reduced in the presence of serum, inferring the possibility of a native inhibitory ligand. Homology modelling and ligand docking studies led to the identification of a novel compound that inhibited GPR21 activity. Its effects offer potential as an anti-diabetic pharmacological strategy as it was found to counteract the influence of GPR21 on the insulin signalling pathway.

  13. Vitamin D and alternative splicing of RNA.

    Science.gov (United States)

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Genetic deletion of GPR52 enhances the locomotor-stimulating effect of an adenosine A2A receptor antagonist in mice: A potential role of GPR52 in the function of striatopallidal neurons.

    Science.gov (United States)

    Nishiyama, Keiji; Suzuki, Hirobumi; Maruyama, Minoru; Yoshihara, Tomoki; Ohta, Hiroyuki

    2017-09-01

    G protein-coupled receptor 52 (GPR52) is largely co-expressed with dopamine D 2 receptor (DRD2) in the striatum and nucleus accumbens, and this expression pattern is similar to that of adenosine A 2A receptor (ADORA2A). GPR52 has been proposed as a therapeutic target for positive symptoms of schizophrenia, based on observations from pharmacological and transgenic mouse studies. However, the physiological role of GPR52 in dopaminergic functions in the basal ganglia remains unclear. Here, we used GPR52 knockout (KO) mice to examine the role of GPR52 in dopamine receptor-mediated and ADORA2A-mediated locomotor activity and dopamine receptor signaling. High expression of GPR52 protein in the striatum, nucleus accumbens, and lateral globus pallidus of wild type (WT) littermates was confirmed by immunohistochemical analysis. GPR52 KO and WT mice exhibited almost identical locomotor responses to the dopamine releaser methamphetamine and the N-methyl-d-aspartate antagonist MK-801. In contrast, the locomotor response to the ADORA2A antagonist istradefylline was significantly augmented in GPR52 KO mice compared to WT mice. Gene expression analysis revealed that striatal expression of DRD2, but not of dopamine D 1 receptor and ADORA2A, was significantly decreased in GPR52 KO mice. Moreover, a significant reduction in the mRNA expression of enkephalin, a marker of the activity of striatopallidal neurons, was observed in the striatum of GPR52 KO mice, suggesting that GPR52 deletion could enhance DRD2 signaling. Taken together, these results imply the physiological relevance of GPR52 in modulating the function of striatopallidal neurons, possibly by interaction of GPR52 with ADORA2A and DRD2. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Signaling via G proteins mediates tumorigenic effects of GPR87

    DEFF Research Database (Denmark)

    Arfelt, Kristine Niss; Fares, Suzan; Sparre-Ulrich, Alexander H.

    2017-01-01

    G protein-coupled receptors (GPCRs) constitute a large protein family of seven transmembrane (7TM) spanning proteins that regulate multiple physiological functions. GPR87 is overexpressed in several cancers and plays a role in tumor cell survival. Here, the basal activity of GPR87 was investigated...

  16. GPR utilization in artificial freezing engineering

    International Nuclear Information System (INIS)

    Song, Lei; Yang, Weihao; Huang, Jiahui; Li, Haipeng; Zhang, Xiaojun

    2013-01-01

    To utilize ground penetrating radar (GPR) in artificial freezing engineering (AFE), the electromagnetic parameters (EMP) of frozen soil were measured using a vector network analyser, which showed that the dielectric permittivity and electric conductivity change abruptly at the boundary between the frozen and the non-frozen soil. Then similarity criteria of GPR model experiments were deduced, and GPR laboratory model experiments and field explorations of AFE were carried out. It was found that for AFE, the GPR travel time and profile characters of anomalies in model experiments were similar to those in field explorations, while the amplitude of GPR signals in laboratory model experiments were much stronger than those in field explorations. Numerical simulations were also implemented to analyse the relationship between model experiments and field explorations, which further told us why we could easily find the targets by GPR in the laboratory but not in field explorations. The outputs showed that GPR could be used to detect the thickness of the frozen wall and to find unfrozen soil defects, even though the amplitude of the reflective signals were much weaker than those of laboratory experiments. The research findings have an important theoretical value for AFE and permafrost region engineering, and the deduced GPR similarity criteria could be widely used in other GPR model experiments. (paper)

  17. Remodeling of Sensorimotor Brain Connectivity in Gpr88-Deficient Mice.

    Science.gov (United States)

    Arefin, Tanzil Mahmud; Mechling, Anna E; Meirsman, Aura Carole; Bienert, Thomas; Hübner, Neele Saskia; Lee, Hsu-Lei; Ben Hamida, Sami; Ehrlich, Aliza; Roquet, Dan; Hennig, Jürgen; von Elverfeldt, Dominik; Kieffer, Brigitte Lina; Harsan, Laura-Adela

    2017-10-01

    Recent studies have demonstrated that orchestrated gene activity and expression support synchronous activity of brain networks. However, there is a paucity of information on the consequences of single gene function on overall brain functional organization and connectivity and how this translates at the behavioral level. In this study, we combined mouse mutagenesis with functional and structural magnetic resonance imaging (MRI) to determine whether targeted inactivation of a single gene would modify whole-brain connectivity in live animals. The targeted gene encodes GPR88 (G protein-coupled receptor 88), an orphan G protein-coupled receptor enriched in the striatum and previously linked to behavioral traits relevant to neuropsychiatric disorders. Connectivity analysis of Gpr88-deficient mice revealed extensive remodeling of intracortical and cortico-subcortical networks. Most prominent modifications were observed at the level of retrosplenial cortex connectivity, central to the default mode network (DMN) whose alteration is considered a hallmark of many psychiatric conditions. Next, somatosensory and motor cortical networks were most affected. These modifications directly relate to sensorimotor gating deficiency reported in mutant animals and also likely underlie their hyperactivity phenotype. Finally, we identified alterations within hippocampal and dorsal striatum functional connectivity, most relevant to a specific learning deficit that we previously reported in Gpr88 -/- animals. In addition, amygdala connectivity with cortex and striatum was weakened, perhaps underlying the risk-taking behavior of these animals. This is the first evidence demonstrating that GPR88 activity shapes the mouse brain functional and structural connectome. The concordance between connectivity alterations and behavior deficits observed in Gpr88-deficient mice suggests a role for GPR88 in brain communication.

  18. Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

    Directory of Open Access Journals (Sweden)

    Ayaka Tobo

    Full Text Available G protein-coupled receptor 4 (GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

  19. Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

    Science.gov (United States)

    Tobo, Ayaka; Tobo, Masayuki; Nakakura, Takashi; Ebara, Masashi; Tomura, Hideaki; Mogi, Chihiro; Im, Dong-Soon; Murata, Naoya; Kuwabara, Atsushi; Ito, Saki; Fukuda, Hayato; Arisawa, Mitsuhiro; Shuto, Satoshi; Nakaya, Michio; Kurose, Hitoshi; Sato, Koichi; Okajima, Fumikazu

    2015-01-01

    G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

  20. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  1. G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

    Science.gov (United States)

    Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2017-06-16

    G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of G i/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that G i/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two G i/o -mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Deletion of the pH sensor GPR4 decreases renal acid excretion.

    Science.gov (United States)

    Sun, Xuming; Yang, Li V; Tiegs, Brian C; Arend, Lois J; McGraw, Dennis W; Penn, Raymond B; Petrovic, Snezana

    2010-10-01

    Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

  3. Baicalein, unlike 4-hydroxytamoxifen but similar to G15, suppresses 17β-estradiol-induced cell invasion, and matrix metalloproteinase-9 expression and activation in MCF-7 human breast cancer cells.

    Science.gov (United States)

    Chen, Yan; Hong, Duan-Yang; Wang, Jing; Ling-Hu, Jun; Zhang, Yan-Yan; Pan, Di; Xu, Yi-Ni; Tao, Ling; Luo, Hong; Shen, Xiang-Chun

    2017-08-01

    Estrogen performs an important role in the growth and development of breast cancer. There are at least three major receptors, including estrogen receptor (ER)α and β, and G protein-coupled receptor 30 (GPR30), which mediate the actions of estrogen through using transcriptional and rapid non-genomic signaling pathways. Flavonoids have been considered candidates for chemopreventive agents in breast cancer. Baicalein, the primary flavonoid derived from the root of Scutellaria baicalensis Georgi, has been reported to exert an anti-estrogenic effect. In the present study, the effects of baicalein on 17β-estradiol (E2)-induced cell invasion, and matrix metalloproteinase-9 (MMP-9) expression and activation were investigated. Furthermore, its effects were compared with that of the active form of the ER modulator tamoxifen 4-hydroxytamoxifen (OHT) and the GPR30 antagonist G15 in ERα- and GPR30-positive MCF-7 breast cancer cells. The results demonstrated that OHT failed to prevent E2-induced cell invasion, upregulation and proteolytic activity of MMP-9. However, baicalein was able to significantly suppress these E2-induced effects. Furthermore, E2-stimulated invasion, and MMP-9 expression and activation were significantly attenuated following G15 treatment. In addition, baicalein significantly inhibited G-1, a specific GPR30 agonist, induced invasion, and reduced G-1 promoted expression and activity of MMP-9, consistent with effects of G15. The results of the present study suggest that baicalein is a therapeutic candidate for GPR30-positive breast cancer treatment, and besides ERα targeting the GPR30 receptor it may achieve additional therapeutic benefits in breast cancer.

  4. E2GPR - Edit your geometry, Execute GprMax2D and Plot the Results!

    Science.gov (United States)

    Pirrone, Daniele; Pajewski, Lara

    2015-04-01

    In order to predict correctly the Ground Penetrating Radar (GPR) response from a particular scenario, Maxwell's equations have to be solved, subject to the physical and geometrical properties of the considered problem and to its initial conditions. Several techniques have been developed in computational electromagnetics, for the solution of Maxwell's equations. These methods can be classified into two main categories: differential and integral equation solvers, which can be implemented in the time or spectral domain. All of the different methods present compromises between computational efficiency, stability, and the ability to model complex geometries. The Finite-Difference Time-Domain (FDTD) technique has several advantages over alternative approaches: it has inherent simplicity, efficiency and conditional stability; it is suitable to treat impulsive behavior of the electromagnetic field and can provide either ultra-wideband temporal waveforms or the sinusoidal steady-state response at any frequency within the excitation spectrum; it is accurate and highly versatile; and it has become a mature and well-researched technique. Moreover, the FDTD technique is suitable to be executed on parallel-processing CPU-based computers and to exploit the modern computer visualisation capabilities. GprMax [1] is a very well-known and largely validated FDTD software tool, implemented by A. Giannopoulos and available for free public download on www.gprmax.com, together with examples and a detailled user guide. The tool includes two electromagnetic wave simulators, GprMax2D and GprMax3D, for the full-wave simulation of two-dimensional and three-dimensional GPR models. In GprMax, everything can be done with the aid of simple commands that are used to define the model parameters and results to be calculated. These commands need to be entered in a simple ASCII text file. GprMax output files can be stored in ASCII or binary format. The software is provided with MATLAB functions, which

  5. COST Action TU1208 - Working Group 4 - Combined use of GPR and other NDT methods & GPR applications in geosciences

    Science.gov (United States)

    Pajewski, Lara; Solla, Mercedes; Fontul, Simona

    2017-04-01

    courses. Additionally, WG4 was very active in offering training activities. In cooperation with the other WGs, the following courses were successfully organised: Training School (TS) "Civil engineering applications of Ground Penetrating Radar" (Pisa, Italy, September 2014), TS "Applications of Ground Penetrating Radar in urban areas: the sensitive case of historical cities" (Cracow, Poland, May 2015), TS "Applications of GPR to civil engineering and archaeology" (Valletta, Malta, January 2016), and TS "Non-destructive testing techniques for civil engineering" (Barcelona, Spain, March 2016). Finally, WG4 contributed to the organization of a series of national events devoted to fostering the interaction of Action Members with stakeholders, new potential GPR end-users, and interested citizens. During such events, participants could discover what is Ground Penetrating Radar (GPR) and how this technique can be effectively used in civil engineering works as well as in different fields ("TU1208 GPR Road Show"). Acknowledgement: The Authors are deeply grateful to COST (European Cooperation in Science and Technology, www.cost.eu), for funding and supporting the COST Action TU1208 "Civil engineering applications of Ground Penetrating Radar" (www.GPRadar.eu).

  6. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  7. GPR142 Controls Tryptophan-Induced Insulin and Incretin Hormone Secretion to Improve Glucose Metabolism.

    Directory of Open Access Journals (Sweden)

    Hua V Lin

    Full Text Available GPR142, a putative amino acid receptor, is expressed in pancreatic islets and the gastrointestinal tract, but the ligand affinity and physiological role of this receptor remain obscure. In this study, we show that in addition to L-Tryptophan, GPR142 signaling is also activated by L-Phenylalanine but not by other naturally occurring amino acids. Furthermore, we show that Tryptophan and a synthetic GPR142 agonist increase insulin and incretin hormones and improve glucose disposal in mice in a GPR142-dependent manner. In contrast, Phenylalanine improves in vivo glucose disposal independently of GPR142. Noteworthy, refeeding-induced elevations in insulin and glucose-dependent insulinotropic polypeptide are blunted in Gpr142 null mice. In conclusion, these findings demonstrate GPR142 is a Tryptophan receptor critically required for insulin and incretin hormone regulation and suggest GPR142 agonists may be effective therapies that leverage amino acid sensing pathways for the treatment of type 2 diabetes.

  8. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    Directory of Open Access Journals (Sweden)

    Adam L Martin

    Full Text Available The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1 200% elevation over baseline reporter gene expression; 2 40% inhibition of baseline expression; and 3 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1 inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176; 2 no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119; 3 elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12; 4 elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65; and 5 no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87. Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40. This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65% than stimulation of expression (15%. Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

  9. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    Science.gov (United States)

    Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

  10. Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

    Science.gov (United States)

    Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

    2014-01-01

    The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

  11. Bidirectional GPR119 agonism requires peptide YY and glucose for activity in mouse and human colon mucosa

    DEFF Research Database (Denmark)

    Tough, Iain R; Forbes, Sarah; Herzog, Herbert

    2018-01-01

    motility in wild-type (WT), GPR119-/- and PYY-/- mice.The water-soluble GPR119 agonist, AR440006 (that cannot traverse epithelial tight-junctions) elicited responses when added apically or basolaterally in mouse and human colonic mucosas. In both species, GPR119 responses were PYY, Y1 receptor...

  12. GPR120

    DEFF Research Database (Denmark)

    Gao, B.; Huang, Q.; Jie, Q.

    2015-01-01

    /2 cascade, while low concentrations elevated P38 and increased adipogenesis. The fine molecular regulation of GPR120 was implemented by up-regulating different integrin subunits (alpha 1, alpha 2 and beta 1; alpha 5 and beta 3). The administration of high doses of TUG-891 rescued oestrogen-deficient bone...... was involved in the cytoplasmic regulation of a seesaw-like balance between ERK and p38 phosphorylation. These findings provide new hope for developing novel remedies to treat osteoporosis by adjusting the GPR120-mediated differentiation balance of BMMSCs....

  13. GPR Diagnostics of columns in archaeological contexts

    Science.gov (United States)

    Soldovieri, Francesco; Masini, Nicola; Persico, Raffaele; Catapano, Ilaria

    2017-04-01

    In the last decade the use of Ground Penetrating radar (GPR) applied to cultural heritage has been strongly increasing thanks to both technological development of sensors and softwares for data processing and cultural reasons such as the increasing awareness of conservators and archaeologist of the benefits of this method in terms of reduction of costs and time and risk associated with restoration works. This made GPR a mature technique for investigating different types of works of art and building elements of historical interest, including masonry structures, frescoes, mosaics [1-3], in the context of scientific projects, decision support activities aimed at the diagnosis of decay pathologies, and educational activities. One of the most complex building elements to be investigated by GPR are the columns both for the geometry of the object and for the several expected features to be detected including fractures, dishomogeneities and metallic connection elements. The work deals with the Ground Penetrating Radar diagnostic surveys at the prestigious archaeological site of Pompei. In particular, GPR surveys were carried out in two different areas, Palestra Grande and Tempio di Giove. The first campaign was carried out also as educational activity of the "International School "GEOPHYSICS AND REMOTE SENSING FOR ARCHAEOLOGY". The School aimed at giving the opportunity to scholars, PhD students, researchers and specialists in Geophysics, Remote Sensing and Archaeology to deepen their knowledge and expertise with geophysical and remote sensing techniques for archaeology and cultural heritage documentation and management. This survey was carried on two kinds of columns, with circular and rectangular section in order to detect possible hidden defects affecting their integrity. The second survey was carried out at Tempio di Giove, on request of the Soprintendenza Pompei, in order to gain information about the presence of reinforcement structures, which may be put inside the

  14. CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

    Science.gov (United States)

    Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aurélia; Landsberger, Nicoletta; Broccoli, Vania

    2009-12-01

    Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

  15. Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.

    Science.gov (United States)

    Wang, Jinghong; Wu, Xiaosu; Simonavicius, Nicole; Tian, Hui; Ling, Lei

    2006-11-10

    Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.

  16. GPR119 as a fat sensor

    DEFF Research Database (Denmark)

    Hansen, Harald S; Rosenkilde, Mette M; Holst, Jens Juul

    2012-01-01

    acting through, for example, GPR40, but is also probably mediated in large part through the luminal formation of 2-monoacylglycerol acting on the 'fat sensor' GPR119. In the pancreas GPR119 may also be stimulated by 2-monoacylglycerol generated from local turnover of pancreatic triacylglycerol. Knowledge...

  17. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    International Nuclear Information System (INIS)

    Zhao, Zhuo; Wang, Hao; Lin, Marina; Groban, Leanne

    2015-01-01

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression

  18. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Zhuo [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013 (China); Wang, Hao; Lin, Marina [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Groban, Leanne, E-mail: lgroban@wakehealth.edu [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Hypertension and Vascular Disease Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States); Office of Women in Medicine and Science, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States)

    2015-03-27

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.

  19. Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets.

    Science.gov (United States)

    Ulven, Trond

    2012-01-01

    The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD). Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and in regulation of appetite. More research is however required to clarify the potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets.

  20. Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets

    Directory of Open Access Journals (Sweden)

    Trond eUlven

    2012-10-01

    Full Text Available The deorphanization of the free fatty acid (FFA receptors FFA1 (GPR40, FFA2 (GPR43, FFA3 (GPR41, GPR84 and GPR120 made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane receptors free fatty acid receptor 2 (FFA2 and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD. Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and regulation of appetite. More research is however required to clarify potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that the situation may soon improve, and that proper tool compounds will enable studies critical to validate the receptors as new drug targets.

  1. Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.

    Directory of Open Access Journals (Sweden)

    Sushma Grellscheid

    2011-12-01

    Full Text Available Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10 is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl; Nestin-Cre(tg/+. This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.

  2. Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R.

    Science.gov (United States)

    Sundqvist, Martina; Christenson, Karin; Holdfeldt, André; Gabl, Michael; Mårtensson, Jonas; Björkman, Lena; Dieckmann, Regis; Dahlgren, Claes; Forsman, Huamei

    2018-05-01

    GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. GPR119 - a major Enteroendocrine Sensor of Dietary Triglyceride Metabolites Co-acting in Synergy with FFA1 (GPR40)

    DEFF Research Database (Denmark)

    Ekberg, Jeppe H; Pedersen, Maria Hauge; Kristensen, Line V

    2016-01-01

    the FFA1 agonist was administered on top of appropriately low doses of the GPR119 agonist a clear synergistic, i.e. more than additive effect was observed. It is concluded that the 2-MAG receptor GPR119 is at least as important as the LCFA receptor FFA1 in mediating the triglyceride-induced secretion......Triglycerides are among the most efficacious stimulators of incretin secretion; however the relative importance of FFA1 (GPR40), FFA4 (GPR120) and GPR119 which all recognize triglyceride metabolites, i.e. LCFA and 2-MAG respectively, is still unclear. Here, we find all three receptors to be highly...... the synthetic FFA1 agonists, TAK-875 stimulated GLP-1 secretion to a similar extent as the prototype GLP-1 secretagogue neuromedin C, this however only corresponded to approx. half the maximal efficiency of the GPR119 agonist AR231453 whereas the GPR120 agonist Metabolix-209 had no effect. Importantly, when...

  4. Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

    Science.gov (United States)

    Konno, Yasunori; Ueki, Shigeharu; Takeda, Masahide; Kobayashi, Yoshiki; Tamaki, Mami; Moritoki, Yuki; Oyamada, Hajime; Itoga, Masamichi; Kayaba, Hiroyuki; Omokawa, Ayumi; Hirokawa, Makoto

    2015-01-01

    Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

  5. GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine

    Directory of Open Access Journals (Sweden)

    David B. Finlay

    2016-03-01

    Full Text Available The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly. Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP and phosphorylation of extracellular signal-regulated kinase (ERK, in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R, were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine. This A108N mutant

  6. The GPR139 reference agonists 1a and 7c, and tryptophan and phenylalanine share a common binding site

    DEFF Research Database (Denmark)

    Shehata, Mohamed A.; Jensen, Anne Cathrine Nøhr; Jespers, Willem

    2017-01-01

    for GPR139. Two series published by Shi et al. and Dvorak et al. included agonists 1a and 7c respectively, with potencies in the ten-nanomolar range. Furthermore, Isberg et al. and Liu et al. have previously shown that tryptophan (Trp) and phenylalanine (Phe) can activate GPR139 in the hundred...... important for the activation of GPR139 as predicted by the receptor model. The initial ligand-receptor complex was optimized through free energy perturbation simulations, generating a refined GPR139 model in agreement with experimental data. In summary, the GPR139 reference surrogate agonists 1a and 7c...

  7. GPR in Ramboll

    Science.gov (United States)

    Ringgaard, Jørgen; Wisén, Roger

    2014-05-01

    The Ramboll Group is a large (10.000 employees worldwide) engineering and consultancy company, with offices in 21 countries. Ramboll has been working with geophysics for about 20 years and at the time of writing there are about 25 geophysicist employed in the group, 20 of these are employed in Ramboll Denmark. Ramboll offers an extensive range of geophysical methods: different types of seismic, borehole wireline logging, electric and electromagnetic surveys, magnetic resonance soundings and well as marine geophysical and hydrographic surveys. The geophysical group at Ramboll operates in different industries comprising: Infrastructure, environmental assessments, mineral exploration, energy and offshore constructions. In the recent years our GPR activities has increased significantly. Today Ramboll Denmark owns three separate GPR systems: One GSSI SIR-3000 with antennas ranging from 16MHz to 2GHz, One Mala geoscience ProEx system with a 100MHz RTA antenna and one 3D-radar Geoscope MKIV system with two DX antennas of different size. The main services are geological mapping with our ProEx system from Malå Geoscience, road mapping with a GSSI system and different shallow mapping with our 3D system from 3D Radar. With our 2D systems we have performed mapping of peat in different places in Norway, mapping of sediments at various places in the Nordic countries and mapping of glacier thickness in Greenland. In this type of investigations we often combine GPR with resistivity imaging (CVES) and refraction seismic to ensure a more reliable interpretation. We have performed occasional utility or UXO surveys where GPR has been used together with EM or magnetic measurements. The mapping on roads with the GSSI system is performed by our RST (Road Surface Testing) department in Malmö, Sweden. The measurements on roads are often combined with laser scanning and photo registration of the surface. Various software have been developed to automatize the interpretation. The RST group

  8. The connection between splicing and cancer

    OpenAIRE

    Srebrow, Anabella; Kornblihtt, Alberto Rodolfo

    2017-01-01

    Alternative splicing is a crucial mechanism for generating protein diversity. Different splice variants of a given protein can display different and even antagonistic biological functions. Therefore, appropriate control of their synthesis is required to assure the complex orchestration of cellular processes within multicellular organisms. Mutations in cisacting splicing elements or changes in the activity of regulatory proteins that compromise the accuracy of either constitutive or alternativ...

  9. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

    Science.gov (United States)

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-01-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  10. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells

    Directory of Open Access Journals (Sweden)

    Gin-Wen Chang

    2016-05-01

    Full Text Available Natural killer (NK cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

  11. GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte

    2015-01-01

    Background: Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. Methods: In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail...... investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. Conclusions: There were no changes in the GPR39 knockout...

  12. Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor.

    Science.gov (United States)

    Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

    2013-04-12

    G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [(35)S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease.

  13. Numerical Study of Planar GPR Antenna Measurements

    DEFF Research Database (Denmark)

    Meincke, Peter; Hansen, Thorkild

    2004-01-01

    The formulation of planar near-field measurements of GPR antennas determines the plane-wave spectra of the GPR antenna in terms of measurements obtained with a buried probe as the GPR antenna moves over a scan plane on the ground. A numerical study investigates how the formulation is affected by (1...

  14. Modelling reveals kinetic advantages of co-transcriptional splicing.

    Directory of Open Access Journals (Sweden)

    Stuart Aitken

    2011-10-01

    Full Text Available Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

  15. Modelling reveals kinetic advantages of co-transcriptional splicing.

    Science.gov (United States)

    Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

    2011-10-01

    Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

  16. The GPR120 agonist TUG-891 promotes metabolic health by stimulating mitochondrial respiration in brown fat

    DEFF Research Database (Denmark)

    Schilperoort, Maaike; van Dam, Andrea D; Hoeke, Geerte

    2018-01-01

    the therapeutic potential of GPR120 agonism and addressed GPR120-mediated signaling in BAT We found that activation of GPR120 by the selective agonist TUG-891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content...

  17. Assessment of highway pavements using GPR

    Science.gov (United States)

    Plati, Christina; Loizos, Andreas

    2015-04-01

    Highway infrastructure is a prerequisite for a functioning economy and social life. Highways, often prone to congestion and disruption, are one of the aspects of a modern transport network that require maximum efficiency if an integrated transport network, and sustainable mobility, is to be achieved. Assessing the condition of highway structures, to plan subsequent maintenance, is essential to allow the long-term functioning of a road network. Optimizing the methods used for such assessment will lead to better information being obtained about the road and underlying ground conditions. The condition of highway structures will be affected by a number of factors, including the properties of the highway pavement, the supporting sub-base and the subgrade (natural ground), and the ability to obtain good information about the entire road structure, from pavement to subgrade, allows appropriate maintenance programs to be planned. The maintenance of highway pavements causes considerable cost and in many cases obstruction to traffic flow. In this situation, methods that provide information on the present condition of pavement structure non-destructively and economically are of great interest. It has been shown that Ground-Penetrating-Radar (GPR), which is a Non Destructive Technique (NDT), can deliver information that is useful for the planning of pavement maintenance activities. More specifically GPR is used by pavement engineers in order to determine physical properties and characteristics of the pavement structure, information that is valuable for the assessment of pavement condition. This work gives an overview on the practical application of GPR using examples from highway asphalt pavements monitoring. The presented individual applications of GPR pavement diagnostics concern structure homogeneity, thickness of pavement layers, dielectric properties of asphalt materials etc. It is worthwhile mentioning that a number of applications are standard procedures, either

  18. GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

    Directory of Open Access Journals (Sweden)

    Chia-Shan Wu

    Full Text Available The G-protein coupled receptor 55 (GPR55 is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

  19. GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

    Science.gov (United States)

    Wu, Chia-Shan; Chen, Hongmei; Sun, Hao; Zhu, Jie; Jew, Chris P; Wager-Miller, James; Straiker, Alex; Spencer, Corinne; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2013-01-01

    The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

  20. Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation

    DEFF Research Database (Denmark)

    Dragano, Nathalia R V; Solon, Carina; Ramalho, Albina F

    2017-01-01

    BACKGROUND: The consumption of large amounts of dietary fats is one of the most important environmental factors contributing to the development of obesity and metabolic disorders. GPR120 and GPR40 are polyunsaturated fatty acid receptors that exert a number of systemic effects that are beneficial...... for metabolic and inflammatory diseases. Here, we evaluate the expression and potential role of hypothalamic GPR120 and GPR40 as targets for the treatment of obesity. METHODS: Male Swiss (6-weeks old), were fed with a high fat diet (HFD, 60% of kcal from fat) for 4 weeks. Next, mice underwent stereotaxic...... the treatment period. At the end of the experiment, the hypothalamus was collected for real-time PCR analysis. RESULTS: We show that both receptors are expressed in the hypothalamus; GPR120 is primarily present in microglia, whereas GPR40 is expressed in neurons. Upon intracerebroventricular treatment, GW9508...

  1. Progress in quantitative GPR development at CNDE

    Science.gov (United States)

    Eisenmann, David; Margetan, F. J.; Chiou, C.-P.; Roberts, Ron; Wendt, Scott

    2014-02-01

    Ground penetrating radar (GPR) uses electromagnetic (EM) radiation pulses to locate and map embedded objects. Commercial GPR instruments are generally geared toward producing images showing the location and extent of buried objects, and often do not make full use of available absolute amplitude information. At the Center for Nondestructive Evaluation (CNDE) at Iowa State University efforts are underway to develop a more quantitative approach to GPR inspections in which absolute amplitudes and spectra of measured signals play a key role. Guided by analogous work in ultrasonic inspection, there are three main thrusts to the effort. These focus, respectively, on the development of tools for: (1) analyzing raw GPR data; (2) measuring the EM properties of soils and other embedding media; and (3) simulating GPR inspections. This paper reviews progress in each category. The ultimate goal of the work is to develop model-based simulation tools that can be used assess the usefulness of GPR for a given inspection scenario, to optimize inspection choices, and to determine inspection reliability.

  2. Progress in quantitative GPR development at CNDE

    International Nuclear Information System (INIS)

    Eisenmann, David; Margetan, F. J.; Chiou, C.-P.; Roberts, Ron; Wendt, Scott

    2014-01-01

    Ground penetrating radar (GPR) uses electromagnetic (EM) radiation pulses to locate and map embedded objects. Commercial GPR instruments are generally geared toward producing images showing the location and extent of buried objects, and often do not make full use of available absolute amplitude information. At the Center for Nondestructive Evaluation (CNDE) at Iowa State University efforts are underway to develop a more quantitative approach to GPR inspections in which absolute amplitudes and spectra of measured signals play a key role. Guided by analogous work in ultrasonic inspection, there are three main thrusts to the effort. These focus, respectively, on the development of tools for: (1) analyzing raw GPR data; (2) measuring the EM properties of soils and other embedding media; and (3) simulating GPR inspections. This paper reviews progress in each category. The ultimate goal of the work is to develop model-based simulation tools that can be used assess the usefulness of GPR for a given inspection scenario, to optimize inspection choices, and to determine inspection reliability

  3. Progress in quantitative GPR development at CNDE

    Energy Technology Data Exchange (ETDEWEB)

    Eisenmann, David; Margetan, F. J.; Chiou, C.-P.; Roberts, Ron; Wendt, Scott [Center for Nondestructive Evaluation, Iowa State University, 1915 Scholl Road, Ames, IA 50011-3042 (United States)

    2014-02-18

    Ground penetrating radar (GPR) uses electromagnetic (EM) radiation pulses to locate and map embedded objects. Commercial GPR instruments are generally geared toward producing images showing the location and extent of buried objects, and often do not make full use of available absolute amplitude information. At the Center for Nondestructive Evaluation (CNDE) at Iowa State University efforts are underway to develop a more quantitative approach to GPR inspections in which absolute amplitudes and spectra of measured signals play a key role. Guided by analogous work in ultrasonic inspection, there are three main thrusts to the effort. These focus, respectively, on the development of tools for: (1) analyzing raw GPR data; (2) measuring the EM properties of soils and other embedding media; and (3) simulating GPR inspections. This paper reviews progress in each category. The ultimate goal of the work is to develop model-based simulation tools that can be used assess the usefulness of GPR for a given inspection scenario, to optimize inspection choices, and to determine inspection reliability.

  4. Mutation analysis and molecular modeling for the investigation of ligand-binding modes of GPR84.

    Science.gov (United States)

    Nikaido, Yoshiaki; Koyama, Yuuta; Yoshikawa, Yasushi; Furuya, Toshio; Takeda, Shigeki

    2015-05-01

    GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology model of a GPR84-capric acid complex to investigate the ligand-binding mode using the crystal structure of human active-state β2-adrenergic receptor. We performed site-directed mutagenesis to subject ligand-binding sites to our model using GPR84-Giα fusion proteins and a [(35)S]GTPγS-binding assay. We compared the activity of the wild type and mutated forms of GPR84 by [(35)S]GTPγS binding to capric acid and diindolylmethane. The mutations L100D `Ballesteros-Weinstein numbering: 3.32), F101Y (3.33) and N104Q (3.36) in the transmembrane helix III and N357D (7.39) in the transmembrane helix VII resulted in reduced capric acid activity but maintained the diindolylmethane responses. Y186F (5.46) and Y186H (5.46) mutations had no characteristic effect on capric acid but with diindolylmethane they significantly affected the G protein activation efficiency. The L100D (3.32) mutant responded to decylamine, a fatty amine, instead of a natural agonist, the fatty acid capric acid, suggesting that we have identified a mutated G protein-coupled receptor-artificial ligand pairing. Our molecular model provides an explanation for these results and interactions between GPR84 and capric acid. Further, from the results of a double stimulation assay, we concluded that diindolylmethane was a positive allosteric modulator for GPR84. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  5. Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92*S⃞

    OpenAIRE

    Oh, Da Young; Yoon, Jung Min; Moon, Mi Jin; Hwang, Jong-Ik; Choe, Han; Lee, Ju Yeon; Kim, Jae Il; Kim, Sunoh; Rhim, Hyewhon; O'Dell, David K.; Walker, J. Michael; Na, Heung Sik; Lee, Min Goo; Kwon, Hyuk Bang; Kim, Kyungjin

    2008-01-01

    A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both Gq/11- and Gs-mediated signaling pathways, whereas NAG activated only the Gq/11-mediated signaling pathway. Co...

  6. Alternative Splicing in Neurogenesis and Brain Development.

    Science.gov (United States)

    Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

    2018-01-01

    Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  7. Alternative Splicing in Neurogenesis and Brain Development

    Directory of Open Access Journals (Sweden)

    Chun-Hao Su

    2018-02-01

    Full Text Available Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  8. Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

    Science.gov (United States)

    De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

    2016-01-01

    Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

  9. Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells.

    Directory of Open Access Journals (Sweden)

    Katie M Lowther

    Full Text Available G protein-coupled receptor 3 (GPR3 is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2 and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third

  10. GPR68 Senses Flow and Is Essential for Vascular Physiology.

    Science.gov (United States)

    Xu, Jie; Mathur, Jayanti; Vessières, Emilie; Hammack, Scott; Nonomura, Keiko; Favre, Julie; Grimaud, Linda; Petrus, Matt; Francisco, Allain; Li, Jingyuan; Lee, Van; Xiang, Fu-Li; Mainquist, James K; Cahalan, Stuart M; Orth, Anthony P; Walker, John R; Ma, Shang; Lukacs, Viktor; Bordone, Laura; Bandell, Michael; Laffitte, Bryan; Xu, Yan; Chien, Shu; Henrion, Daniel; Patapoutian, Ardem

    2018-04-19

    Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. GPR142 Controls Tryptophan-Induced Insulin and Incretin Hormone Secretion to Improve Glucose Metabolism

    OpenAIRE

    Lin, Hua V.; Efanov, Alexander M.; Fang, Xiankang; Beavers, Lisa S.; Wang, Xuesong; Wang, Jingru; Gonzalez Valcarcel, Isabel C.; Ma, Tianwei

    2016-01-01

    GPR142, a putative amino acid receptor, is expressed in pancreatic islets and the gastrointestinal tract, but the ligand affinity and physiological role of this receptor remain obscure. In this study, we show that in addition to L-Tryptophan, GPR142 signaling is also activated by L-Phenylalanine but not by other naturally occurring amino acids. Furthermore, we show that Tryptophan and a synthetic GPR142 agonist increase insulin and incretin hormones and improve glucose disposal in mice in a G...

  12. Potential roles of GPR120 and its agonists in the management of diabetes

    Directory of Open Access Journals (Sweden)

    Zhang D

    2014-07-01

    Full Text Available Dan Zhang, Po Sing Leung School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Abstract: Free fatty acids (FFAs serve not only as nutrients that provide energy but also as extracellular signaling molecules that manipulate intracellular physiological events through FFA receptors (FFARs such as FFAR4. FFAR4 is also known as G-protein coupled receptor 120 (GPR120. The main role of GPR120 is to elicit FFA regulation on metabolism homeostasis. GPR120 agonism correlates with prevention of the occurrence and development of metabolic disorders such as obesity and diabetes. GPR120 activation directly or indirectly inhibits inflammation, modulates hormone secretion from the gastrointestinal tract and pancreas, and regulates lipid and/or glucose metabolism in adipose, liver, and muscle tissues, which may help prevent obesity and diabetes. This review summarizes recent advances in physiological roles of GPR120 in preventing insulin resistance and protecting pancreatic islet function, and examines how resident GPR120 in the pancreas may be involved in modulating pancreatic islet function. Keywords: fatty acid, G protein-coupled receptors, inflammation, pancreas, obesity

  13. SplicePlot: a utility for visualizing splicing quantitative trait loci.

    Science.gov (United States)

    Wu, Eric; Nance, Tracy; Montgomery, Stephen B

    2014-04-01

    RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available.

  14. Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

    Science.gov (United States)

    Ganay, Thibault; Asraf, Hila; Aizenman, Elias; Bogdanovic, Milos; Sekler, Israel; Hershfinkel, Michal

    2015-12-01

    Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive

  15. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

    Directory of Open Access Journals (Sweden)

    Anne-Mette Hartung

    2016-05-01

    Full Text Available Costello syndrome (CS may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE and creation of an Exonic Splicing Silencer (ESS. We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

  16. GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior

    Directory of Open Access Journals (Sweden)

    Fuka Aizawa

    2016-12-01

    Full Text Available The free fatty acid receptor 1 (GPR40/FFAR1 is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO mice. The emotional behavior in wild and KO male mice was evaluated at 9–10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC–MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain.

  17. IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

    Science.gov (United States)

    Bardhan, Kankana; Paschall, Amy V; Yang, Dafeng; Chen, May R; Simon, Priscilla S; Bhutia, Yangzom D; Martin, Pamela M; Thangaraju, Muthusamy; Browning, Darren D; Ganapathy, Vadivel; Heaton, Christopher M; Gu, Keni; Lee, Jeffrey R; Liu, Kebin

    2015-07-01

    Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. ©2015 American Association for Cancer Research.

  18. IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer

    Science.gov (United States)

    Bardhan, Kankana; Paschall, Amy V.; Yang, Dafeng; Chen, May R.; Simon, Priscilla S.; Bhutia, Yangzom; Martin, Pamela M.; Thangaraju, Muthusamy; Browning, Darren D.; Ganapathy, Vadivel; Heaton, Christopher M.; Gu, Keni; Lee, Jeffrey R.; Liu, Kebin

    2015-01-01

    Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A has been the subject of extensive studies, however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wildtype mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoters to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

  19. A Comprehensive Analysis of Alternative Splicing in Paleopolyploid Maize

    Directory of Open Access Journals (Sweden)

    Wenbin Mei

    2017-05-01

    Full Text Available Identifying and characterizing alternative splicing (AS enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs identified splicing QTL (sQTL. The 43.3% of cis-sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans-sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.

  20. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wan, Lei [Department of Pharmacology, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wang, Xudong, E-mail: xdwang@gmc.edu.cn [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China)

    2014-03-01

    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

  1. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    International Nuclear Information System (INIS)

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia; Wan, Lei; Wang, Xudong

    2014-01-01

    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI

  2. GPR surveying of transport infrastructures and buildings; underground utility and void sensing - ongoing activities in Working Group 2 of COST Action TU1208

    Science.gov (United States)

    Pajewski, Lara; Plati, Christina; Derobert, Xavier

    2015-04-01

    This work aims at presenting the ongoing research activities carried out in Working Group 2 'GPR surveying of pavements, bridges, tunnels and buildings; underground utility and void sensing' of the COST (European COoperation in Science and Technology) Action TU1208 'Civil Engineering Applications of Ground Penetrating Radar' (www.GPRadar.eu). The principal goal of the COST Action TU1208 is to exchange and increase scientific-technical knowledge and experience of Ground Penetrating Radar (GPR) techniques in civil engineering, whilst simultaneously promoting throughout Europe the effective use of this safe and non-destructive technique in the monitoring of infrastructures and structures. Four Working Groups (WGs) carry out the research activities. WG1 focuses on the development of innovative GPR equipment dedicated for civil engineering applications. WG2 deals with the development of guidelines and protocols for the surveying, through the use of a GPR system, of transport infrastructure and buildings, as well as for the sensing of utilities and voids. WG3 deals with the development of electromagnetic forward and inverse scattering methods, for the characterization of GPR scenarios, as well as with data- processing algorithms for the elaboration of the data collected during GPR surveys. WG4 is concerned with the use of GPR in fields different from the civil engineering, as well as with the integration of GPR with other non-destructive testing techniques. Each WG includes several Projects. WG2 includes five Projects. Project 2.1 focuses on outlining 'Innovative inspection procedures for effective GPR surveying of critical transport infrastructures (pavements, bridges and tunnels).' Project 2.2 is concerned with the development of 'Innovative inspection procedures for effective GPR surveying of buildings.' Project 2.3 deals with identifying 'Innovative inspection procedures for effective GPR sensing and mapping of underground utilities and voids, with a focus to urban

  3. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy)

    DEFF Research Database (Denmark)

    Vorwerk, P; Christoffersen, C T; Müller, J

    1999-01-01

    to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase......The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found...... domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing...

  4. Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

    Directory of Open Access Journals (Sweden)

    Julie Carnesecchi

    Full Text Available The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2 addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

  5. Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

    Science.gov (United States)

    Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

    2015-01-01

    The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

  6. GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

    Science.gov (United States)

    Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

    2014-10-31

    Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

  7. Macrophage PPARγ inhibits Gpr132 to mediate the anti-tumor effects of rosiglitazone

    Science.gov (United States)

    Cheng, Wing Yin; Huynh, HoangDinh; Chen, Peiwen; Peña-Llopis, Samuel; Wan, Yihong

    2016-01-01

    Tumor-associated macrophage (TAM) significantly contributes to cancer progression. Human cancer is enhanced by PPARγ loss-of-function mutations, but inhibited by PPARγ agonists such as TZD diabetes drugs including rosiglitazone. However, it remains enigmatic whether and how macrophage contributes to PPARγ tumor-suppressive functions. Here we report that macrophage PPARγ deletion in mice not only exacerbates mammary tumor development but also impairs the anti-tumor effects of rosiglitazone. Mechanistically, we identify Gpr132 as a novel direct PPARγ target in macrophage whose expression is enhanced by PPARγ loss but repressed by PPARγ activation. Functionally, macrophage Gpr132 is pro-inflammatory and pro-tumor. Genetic Gpr132 deletion not only retards inflammation and cancer growth but also abrogates the anti-tumor effects of PPARγ and rosiglitazone. Pharmacological Gpr132 inhibition significantly impedes mammary tumor malignancy. These findings uncover macrophage PPARγ and Gpr132 as critical TAM modulators, new cancer therapeutic targets, and essential mediators of TZD anti-cancer effects. DOI: http://dx.doi.org/10.7554/eLife.18501.001 PMID:27692066

  8. GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior.

    Science.gov (United States)

    Aizawa, Fuka; Nishinaka, Takashi; Yamashita, Takuya; Nakamoto, Kazuo; Kurihara, Takashi; Hirasawa, Akira; Kasuya, Fumiyo; Miyata, Atsuro; Tokuyama, Shogo

    2016-12-01

    The free fatty acid receptor 1 (GPR40/FFAR1) is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO) mice. The emotional behavior in wild and KO male mice was evaluated at 9-10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC-MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  9. Identification of GPR65, a novel regulator of matrix metalloproteinases using high through-put screening

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hongbo; Chen, Xiaohong; Huang, Junwei [Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery, Beijing (China); Deng, Weiwei [Functional Genomics Group, Chinese National Human Genome Center (CHGB) at Beijing (China); Zhong, Qi [Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery, Beijing (China); Yue, Changli [Department of Pathology, Beijing Tongren Hospital, Capital Medical University, Beijing (China); Wang, Pingzhang, E-mail: wangpzh@bjmu.edu.cn [Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Peking University Center for Human Disease Genomics, Key Laboratory of Medical Immunology, Ministry of Health (China); Functional Genomics Group, Chinese National Human Genome Center (CHGB) at Beijing (China); Huang, Zhigang, E-mail: enthuangzhigang@sohu.com [Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery, Beijing (China)

    2013-06-21

    Highlights: •A novel mechanism of MMP3 regulation by proton-sensing G-protein-coupled receptors was defined. •GPR65 was identified to induce the MMP3 expression. •GPR65 mediated MMP induction under acidic conditions. •AP-1 binding site in MMP3 promoter was crucial for MMP3 induction. •GPR65 overexpression can accelerate the invision of A549 cells. -- Abstract: Matrix metalloproteinases (MMPs) are over-expressed in nearly all cancers. To study novel regulatory factors of MMP expression in head and neck cancer (HNC), we screened a total of 636 candidate genes encoding putative human transmembrane proteins using MMP promoter reporter in a dual luciferase assay system. Three genes GPR65, AXL and TNFRSF10B dramatically activated the induction of MMP3 expression. The induction of MMP expression by GPR65 was further confirmed in A549 and/or FaDu cells. GPR65 mediated MMP induction under acidic conditions. The AP-1 binding site in MMP3 promoter was crucial for MMP3 induction. Moreover, the A549 cells infected by recombinant adenovirus of GPR65 showed accelerated cell invasion. In conclusion, we validate that GPR65 is vital regulatory genes upstream of MMP3, and define a novel mechanism of MMP3 regulation by proton-sensing G-protein-coupled receptors.

  10. Identification of GPR65, a novel regulator of matrix metalloproteinases using high through-put screening

    International Nuclear Information System (INIS)

    Xu, Hongbo; Chen, Xiaohong; Huang, Junwei; Deng, Weiwei; Zhong, Qi; Yue, Changli; Wang, Pingzhang; Huang, Zhigang

    2013-01-01

    Highlights: •A novel mechanism of MMP3 regulation by proton-sensing G-protein-coupled receptors was defined. •GPR65 was identified to induce the MMP3 expression. •GPR65 mediated MMP induction under acidic conditions. •AP-1 binding site in MMP3 promoter was crucial for MMP3 induction. •GPR65 overexpression can accelerate the invision of A549 cells. -- Abstract: Matrix metalloproteinases (MMPs) are over-expressed in nearly all cancers. To study novel regulatory factors of MMP expression in head and neck cancer (HNC), we screened a total of 636 candidate genes encoding putative human transmembrane proteins using MMP promoter reporter in a dual luciferase assay system. Three genes GPR65, AXL and TNFRSF10B dramatically activated the induction of MMP3 expression. The induction of MMP expression by GPR65 was further confirmed in A549 and/or FaDu cells. GPR65 mediated MMP induction under acidic conditions. The AP-1 binding site in MMP3 promoter was crucial for MMP3 induction. Moreover, the A549 cells infected by recombinant adenovirus of GPR65 showed accelerated cell invasion. In conclusion, we validate that GPR65 is vital regulatory genes upstream of MMP3, and define a novel mechanism of MMP3 regulation by proton-sensing G-protein-coupled receptors

  11. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  12. Diversification of the muscle proteome through alternative splicing.

    Science.gov (United States)

    Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

    2018-03-06

    Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

  13. A guidelines handbook for GPR surveys in tunnels: a COST Action TU1208 contribution

    Science.gov (United States)

    Bianchini Ciampoli, Luca; Alani, Amir M.; Pajewski, Lara; Benedetto, Andrea; Loizos, Andreas; Tosti, Fabio

    2016-04-01

    A significant open issue concerning the reliability of geophysical methods and in particular of ground penetrating radar (GPR), both in research and professional context, is a general lack of international standards. This is a major problem to be faced, in order to gain scientific strictness for the GPR practices, and to easily extend to the international community the results achieved within the area of single virtuous countries. Producing international guidelines can represent an important step forward, in this sense. In the memorandum of understanding of the COST Action TU1208 is clearly stated that one of the main purposes of the Action is the "development of innovative protocols and guidelines which will be published in a handbook and constitute a basis for European Standards, for an effective GPR application in CE tasks; safety, economic and financial criteria will be integrated within the protocols". Of course this is not a simple task to be accomplished. Firstly, survey procedures are highly dependent on the objective of the survey itself. On the basis of the objective of each geophysical test, the GPR system, the antenna configuration, and even the processing procedures may change. Besides, these procedures are also influenced by the environmental conditions in which the tests are performed. This affects several aspects spanning from hardware to software, but including, for instance, also safety issues. Due to these reasons, one of the main goal of the COST Action TU1208 is the development of several guidelines related to the main applications of GPR in the field of civil engineering. In this work, the structure of a guidelines handbook for GPR activities in tunnels is outlined. In the first sections, the principal references in the field are provided, and the most common GPR equipment and complementary technologies are described. Subsequently, the survey methodologies are explained. Particular attention is paid to the preliminary activities to be carried

  14. Expression and Role of GPR87 in Urothelial Carcinoma of the Bladder

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Kakehi

    2013-06-01

    Full Text Available The orphan GPR87 has recently been matched with its ligand LPA, which is a lipid mediator with multiple physiological functions, including cancer cell proliferation. This study aimed to clarify the role of GPR87 in urothelial carcinoma of the bladder. GPR87 expression was assessed in seven human bladder cancer cell lines. A replication-deficient recombinant adenoviral vector expressing shRNA targeting GPR87 (Ad-shGPR87, was constructed. Gene silencing was carried out using Ad-shGPR87. Immunohistochemical analysis was performed for transurethral resection of bladder tumor samples from 71 patients with non-muscle-invasive bladder cancer. We observed GPR87 expression in five of the seven cell lines, and silencing GPR87 gene expression significantly reduced cell viability. GPR87 expression was positive in 38 (54% of 71 tumors. Ki-67 index was associated with positive GPR87 staining status (p < 0.0001. Patients with GPR87-positive tumors had shorter intravesical recurrence-free survival than those with GPR87-negative tumors (p = 0.010. Multivariate analysis revealed that GPR87 staining status was an independent prognostic parameter for intravesical recurrence (p = 0.041. Progression from non-muscle-invasive to muscle-invasive tumor was more frequently observed in patients with GPR87-positive tumors, although this trend did not reach statistical significance (p = 0.056. These results warrant further prospective studies to clarify the role of GPR87 expression in intravesical recurrence and progression in bladder cancer.

  15. Human Splicing Finder: an online bioinformatics tool to predict splicing signals

    OpenAIRE

    Desmet, Francois-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Beroud, Gwenaelle; Claustres, Mireille; Beroud, Christophe

    2009-01-01

    International audience; Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effec...

  16. Pipe-flange detection with GPR

    International Nuclear Information System (INIS)

    Bonomo, Néstor; De la Vega, Matías; Martinelli, Patricia; Osella, Ana

    2011-01-01

    This paper describes an application of the ground penetrating radar (GPR) method for detecting pipe flanges. A case history is described in which GPR was successfully used to locate pipe flanges along an 8 km metal pipeline, using a fixed-offset methodology, from the ground surface. Summaries of numerical simulations and in situ tests, performed before the definitive prospecting to evaluate the feasibility of detection, are included. Typical GPR signals are analysed and several examples shown. Constant-time sections of data volumes and migration are evaluated with the goal of distinguishing flange signals from rock signals in unclear situations. The applied methodology was effective for detecting the pipe flanges in relatively short times, with accuracies below 10 cm in the horizontal direction and 20 cm in the vertical direction

  17. Neuronal fast activating and meningeal silent modulatory BK channel splice variants cloned from rat

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Jansen-Olesen, Inger; Olesen, Jes

    2011-01-01

    The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned...... and RCK2 (4 aa at SS1) and upstream of the calcium "bowl" (27 aa at SS4). Two other truncated variants, X2(92) and X2(188), lacking the intracellular C-terminal (stop downstream of S6), were cloned from cerebral vascular/meningeal tissue. They appear non-functional as no current expression was observed...

  18. Miniature Ground Penetrating Radar, CRUX GPR

    Science.gov (United States)

    Kim, Soon Sam; Carnes, Steven R.; Haldemann, Albert F.; Ulmer, Christopher T.; Ng, Eddie; Arcone, Steven A.

    2006-01-01

    Under NASA instrument development programs (PIDDP 2000-2002, MIPD 2003-2005, ESR and T, 2005) we have been developing miniature ground penetrating radars (GPR) for use in mapping subsurface stratigraphy from planetary rovers for Mars and lunar applications. The Mars GPR is for deeper penetration (up to 50 m depth) into the Martian subsurface at moderate resolution (0.5 m) for a geological characterization. As a part of the CRUX (Construction and Resource Utilization Explorer) instrument suite, the CRUX GPR is optimized for a lunar prospecting application. It will have shallower penetration (5 m depth) with higher resolution (10 cm) for construction operations including ISRU (in-situ resource utilization).

  19. Antitumorigenic potential of STAT3 alternative splicing modulation.

    Science.gov (United States)

    Zammarchi, Francesca; de Stanchina, Elisa; Bournazou, Eirini; Supakorndej, Teerawit; Martires, Kathryn; Riedel, Elyn; Corben, Adriana D; Bromberg, Jacqueline F; Cartegni, Luca

    2011-10-25

    Signal transducer and activator of transcription 3 (STAT3) plays a central role in the activation of multiple oncogenic pathways. Splicing variant STAT3β uses an alternative acceptor site within exon 23 that leads to a truncated isoform lacking the C-terminal transactivation domain. Depending on the context, STAT3β can act as a dominant-negative regulator of transcription and promote apoptosis. We show that modified antisense oligonucleotides targeted to a splicing enhancer that regulates STAT3 exon 23 alternative splicing specifically promote a shift of expression from STAT3α to STAT3β. Induction of endogenous STAT3β leads to apoptosis and cell-cycle arrest in cell lines with persistent STAT3 tyrosine phosphorylation compared with total STAT3 knockdown obtained by forced splicing-dependent nonsense-mediated decay (FSD-NMD). Comparison of the molecular effects of splicing redirection to STAT3 knockdown reveals a unique STAT3β signature, with a down-regulation of specific targets (including lens epithelium-derived growth factor, p300/CBP-associated factor, CyclinC, peroxisomal biogenesis factor 1, and STAT1β) distinct from canonical STAT3 targets typically associated with total STAT3 knockdown. Furthermore, similar in vivo redirection of STAT3 alternative splicing leads to tumor regression in a xenograft cancer model, demonstrating how pharmacological manipulation of a single key splicing event can manifest powerful antitumorigenic properties and validating endogenous splicing reprogramming as an effective cancer therapeutic approach.

  20. Mechanical rebar splicing

    Directory of Open Access Journals (Sweden)

    Milosavljević Branko

    2014-01-01

    Full Text Available Different mechanical rebar splicing systems are presented, and design situations where mechanical splicing has advantage over reinforcement splicing by overlapping and welding are defined in this paper. New international standards for testing and proof of systems for mechanical rebar splicing quality are considered. Mechanical splicing system for rebar and bolt connection, usable in steel and reinforced concrete structural elements connections, is presented in this paper. There are only few examples of mechanical rebar splicing in our country. The most significant one - the pylon and beam connection at Ada Bridge in Belgrade is presented in the paper. Intensive development of production and use of mechanical rebar splicing systems, research in this area, as well as the publication of international standards prescribing requirements for quality and procedures for proof of quality, represent very good base for development of the corresponding technical norms in Serbia. The legislation in this area would quicken proof of quality procedures, attest and approval issuing for individual products, leading to wider use of this system in all situations where it is in advantage over the classical reinforcement splicing.

  1. GPR 120: The Potential Target for Obesity Treatment.

    Science.gov (United States)

    Tanagho, Peter A; Shohdy, Kyrillus S

    2016-01-01

    G protein coupled receptor 120 (GPR120) is a class of receptors in the gastrointestinal tract (GIT) that is implicated in nutrient sensing and body weight regulation. Functions of GPR120 are thought to be mediated by the release of a group of hormones known as incretins, such as glucagon like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). We have searched PubMed with the keywords "GPR120","GLP-1" and "obesity". Relevant studies were retrieved and included in the review. Recently, many exogenous compounds have been investigated in their role in the release of GLP-1 and in causing weight loss in obese rats. However, some results question the putative role of GPR120 in metabolic homeostasis. Herein, we evaluate the potential use of GPR120 as a target receptor in obesity and found it to be ubiquitous throughout the GIT, with various functions in each site. In order to find the optimal drug, the role of GPR120 in each site needs to be defined and selectivity of the potential drug needs to be studied to ensure the success of this growing line of obesity management.

  2. LSM Proteins Provide Accurate Splicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development[W

    Science.gov (United States)

    Perea-Resa, Carlos; Hernández-Verdeja, Tamara; López-Cobollo, Rosa; Castellano, María del Mar; Salinas, Julio

    2012-01-01

    In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay. PMID:23221597

  3. A novel nonsense mutation of the GPR143 gene identified in a Chinese pedigree with ocular albinism.

    Directory of Open Access Journals (Sweden)

    Naihong Yan

    Full Text Available BACKGROUND: The purpose of this study was to elucidate the molecular basis of ocular albinism type I in a Chinese pedigree. METHODOLOGY/PRINCIPAL FINDINGS: Complete ophthalmologic examinations were performed on 4 patients, 7 carriers and 17 unaffected individuals in this five-generation family. All coding exons of four-point-one (4.1, ezrin, radixin, moesin (FERM domain-containing 7 (FRMD7 and G protein-coupled receptor 143 (GPR143 genes were amplified by polymerase chain reaction (PCR, sequenced and compared with a reference database. Ocular albinism and nystagmus were found in all patients of this family. Macular hypoplasia was present in the patients including the proband. A novel nonsense hemizygous mutation c.807T>A in the GPR143 gene was identified in four patients and the heterozygous mutation was found in seven asymptomatic individuals. This mutation is a substitution of tyrosine for adenine which leads to a premature stop codon at position 269 (p.Y269X of GPR143. CONCLUSIONS/SIGNIFICANCE: This is the first report that p.Y269X mutation of GPR143 gene is responsible for the pathogenesis of familial ocular albinism. These results expand the mutation spectrum of GPR143, and demonstrate the clinical characteristics of ocular albinism type I in Chinese population.

  4. ONKALO EDZ-measurements using ground penetrating radar (GPR) method

    Energy Technology Data Exchange (ETDEWEB)

    Silvast, M.; Wiljanen, B. (Roadscanners Oy, Rovaniemi (Finland))

    2008-09-15

    This report presents pilot project results from various Ground Penetrating Radar (GPR) tests performed on bedrock in ONKALO, the research tunnel system being built for the final disposal of spent nuclear fuel (in Finland). In recent years the GPR technology for structure inspection has improved to faster systems and higher frequencies. Processing and interpretation software has been developed for better visualization of processed data. GPR is a powerful non-destructive testing method with major advantages such as fast measurement speed and continuous survey lines. The purpose of the tests was to determine the capacity of GPR in identifying the Excavation Damaged or Disturbed Zone (EDZ). Topics included comparison of different types of GPR systems and antennas in select locations in the tunnel system and data presentation. High quality GPR data was obtained from all systems that were used on surfaces without concrete or steel reinforcement. Data processed using Geo Doctor software, which enables integrated analysis of available datasets on a single screen, provided promising results. (orig.)

  5. ONKALO EDZ-measurements using ground penetrating radar (GPR) method

    International Nuclear Information System (INIS)

    Silvast, M.; Wiljanen, B.

    2008-09-01

    This report presents pilot project results from various Ground Penetrating Radar (GPR) tests performed on bedrock in ONKALO, the research tunnel system being built for the final disposal of spent nuclear fuel (in Finland). In recent years the GPR technology for structure inspection has improved to faster systems and higher frequencies. Processing and interpretation software has been developed for better visualization of processed data. GPR is a powerful non-destructive testing method with major advantages such as fast measurement speed and continuous survey lines. The purpose of the tests was to determine the capacity of GPR in identifying the Excavation Damaged or Disturbed Zone (EDZ). Topics included comparison of different types of GPR systems and antennas in select locations in the tunnel system and data presentation. High quality GPR data was obtained from all systems that were used on surfaces without concrete or steel reinforcement. Data processed using Geo Doctor software, which enables integrated analysis of available datasets on a single screen, provided promising results. (orig.)

  6. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    Science.gov (United States)

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Expression and functional role of orphan receptor GPR158 in prostate cancer growth and progression.

    Directory of Open Access Journals (Sweden)

    Nitin Patel

    Full Text Available Prostate cancer (PCa is the second-leading cause of cancer-related mortality, after lung cancer, in men from developed countries. In its early stages, primary tumor growth is dependent on androgens, thus generally can be controlled by androgen deprivation therapy (ADT. Eventually however, the disease progresses to castration-resistant prostate cancer (CRPC, a lethal form in need of more effective treatments. G-protein coupled receptors (GPCRs comprise a large clan of cell surface proteins that have been implicated as therapeutic targets in PCa growth and progression. The findings reported here provide intriguing evidence of a role for the newly characterized glutamate family member GPR158 in PCa growth and progression. We found that GPR158 promotes PCa cell proliferation independent of androgen receptor (AR functionality and that this requires its localization in the nucleus of the cell. This suggests that GPR158 acts by mechanisms different from other GPCRs. GPR158 expression is stimulated by androgens and GPR158 stimulates AR expression, implying a potential to sensitize tumors to low androgen conditions during ADT via a positive feedback loop. Further, we found GPR158 expression correlates with a neuroendocrine (NE differentiation phenotype and promotes anchorage-independent colony formation implying a role for GPR158 in therapeutic progression and tumor formation. GPR158 expression was increased at the invading front of prostate tumors that formed in the genetically defined conditional Pten knockout mouse model, and co-localized with elevated AR expression in the cell nucleus. Kaplan-Meier analysis on a dataset from the Memorial Sloan Kettering cancer genome portal showed that increased GPR158 expression in tumors is associated with lower disease-free survival. Our findings strongly suggest that pharmaceuticals targeting GPR158 activities could represent a novel and innovative approach to the prevention and management of CRPC.

  8. Human Splicing Finder: an online bioinformatics tool to predict splicing signals.

    Science.gov (United States)

    Desmet, François-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Béroud, Gwenaëlle; Claustres, Mireille; Béroud, Christophe

    2009-05-01

    Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-beta Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5' and 3' splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.

  9. Modeling of GPR Clutter Caused by Soil Heterogeneity

    Directory of Open Access Journals (Sweden)

    Kazunori Takahashi

    2012-01-01

    Full Text Available In small-scale measurements, ground-penetrating radar (GPR often uses a higher frequency to detect a small object or structural changes in the ground. GPR becomes more sensitive to the natural heterogeneity of the soil when a higher frequency is used. Soil heterogeneity scatters electromagnetic waves, and the scattered waves are in part observed as unwanted reflections that are often referred to as clutter. Data containing a great amount of clutter are difficult to analyze and interpret because clutter disturbs reflections from objects of interest. Therefore, modeling GPR clutter is useful to assess the effectiveness of GPR measurements. In this paper, the development of such a technique is discussed. This modeling technique requires the permittivity distribution of soil (or its geostatistical properties and gives a nominal value of clutter power. The paper demonstrates the technique with the comparison to the data from a GPR time-lapse measurement. The proposed technique is discussed in regard to its applicability and limitations based on the results.

  10. Conditional Toxin Splicing Using a Split Intein System.

    Science.gov (United States)

    Alford, Spencer C; O'Sullivan, Connor; Howard, Perry L

    2017-01-01

    Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

  11. Multiple splicing defects in an intronic false exon.

    Science.gov (United States)

    Sun, H; Chasin, L A

    2000-09-01

    Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

  12. Widespread alternative and aberrant splicing revealed by lariat sequencing

    Science.gov (United States)

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  13. Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes.

    Science.gov (United States)

    Nagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, Yoji

    2012-02-17

    In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  14. Targeting GPR110 in HER2-Overexpressing Breast Cancers

    Science.gov (United States)

    2015-10-01

    34assay"using"the"transwell" inserts"with" (A)"or"without" (B)"matrigel" for"SKBR3" cells "transfected"with"nonAtarge1ng"( NT1 )"orŗ"independent"GPR110"siRNAs...HER2 drug resistant cells and in tumorigenic population using a broad panel of cell line models. We have successfully generated inducible lentiviral...plasmids with GPR110 cDNA and cell lines that inducibly overexpress GPR110. Generation of cell lines with lentiviral plasmids containing GPR110

  15. Ground Penetrating Radar (GPR) for Detection of Underground Objects

    International Nuclear Information System (INIS)

    Amry Amin Abas; Mohd Kamal Shah Shamsuddin; Wan Zainal Abidin; Awang Sarfarudin Awang Putra

    2011-01-01

    Ground Penetrating Radar (GPR) utilizes an electromagnetic microwave that is transmitted into the matter under investigation. Any objects with different dielectric properties from the medium of the matter under investigation will reflect the waves and will be picked up by the receivers embedded in the antenna. We have applied GPR in various application such as concrete inspection, underground utility detection, grave detection, archaeology, oil contamination of soil, soil layer thickness measurement and etc. This paper will give general findings of the application of GPR to provide solutions to the industry and public. The results of the GPR surveys will be discussed. (author)

  16. GPR Imaging of Prehistoric Animal Bone-beds

    Science.gov (United States)

    Schneider, Blair Benson

    This research investigates the detection capabilities of Ground-penetrating radar for imaging prehistoric animal bone-beds. The first step of this investigation was to determine the dielectric properties of modern animal bone as a proxy for applying non-invasive ground-penetrating radar (GPR) for detecting prehistoric animal remains. Over 90 thin section samples were cut from four different modern faunal skeleton remains: bison, cow, deer, and elk. One sample of prehistoric mammoth core was also analyzed. Sample dielectric properties (relative permittivity, loss factor, and loss-tangent values) were measured with an impedance analyzer over frequencies ranging from 10 MHz to 1 GHz. The results reveal statistically significant dielectric-property differences among different animal fauna, as well as variation as a function of frequency. The measured sample permittivity values were then compared to modeled sample permittivity values using common dielectric-mixing models. The dielectric mixing models were used to report out new reported values of dry bone mineral of 3-5 in the frequency range of 10 MHz to 1 GHz. The second half of this research collected controlled GPR experiments over a sandbox containing buried bison bone elements to evaluate GPR detection capabilities of buried animal bone. The results of the controlled GPR sandbox tests were then compared to numerical models in order to predict the ability of GPR to detect buried animal bone given a variety of different depositional factors, the size and orientation of the bone target and the degree of bone weathering. The radar profiles show that GPR is an effective method for imaging the horizontal and vertical extent of buried animal bone. However, increased bone weathering and increased bone dip were both found to affect GPR reflection signal strength. Finally, the controlled sandbox experiments were also utilized to investigate the impact of survey design for imaging buried animal bone. In particular, the

  17. GPR random noise reduction using BPD and EMD

    Science.gov (United States)

    Ostoori, Roya; Goudarzi, Alireza; Oskooi, Behrooz

    2018-04-01

    Ground-penetrating radar (GPR) exploration is a new high-frequency technology that explores near-surface objects and structures accurately. The high-frequency antenna of the GPR system makes it a high-resolution method compared to other geophysical methods. The frequency range of recorded GPR is so wide that random noise recording is inevitable due to acquisition. This kind of noise comes from unknown sources and its correlation to the adjacent traces is nearly zero. This characteristic of random noise along with the higher accuracy of GPR system makes denoising very important for interpretable results. The main objective of this paper is to reduce GPR random noise based on pursuing denoising using empirical mode decomposition. Our results showed that empirical mode decomposition in combination with basis pursuit denoising (BPD) provides satisfactory outputs due to the sifting process compared to the time-domain implementation of the BPD method on both synthetic and real examples. Our results demonstrate that because of the high computational costs, the BPD-empirical mode decomposition technique should only be used for heavily noisy signals.

  18. The application of GPR in survey of Yongshan coalmine bridge

    International Nuclear Information System (INIS)

    Liao Yuhua; Deng Juzhi

    2007-01-01

    Ground penetrating radar (GPR) is an effective shoallow exploration method which uses the reflection of high frequency electromagnetism pulse wave to detect the object and geologic phenomena underground. With the development of electron and computer technique, the application domain of this method is keeping enlarge. Based on the working principle of GPR, this paper discussed the good effect of GPR to pier prospecting in Yongshan coal mines. Some matters that should be attentive are posed while using GPR. (authors)

  19. GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis.

    Science.gov (United States)

    Dietrich, Philipp A; Yang, Chen; Leung, Halina H L; Lynch, Jennifer R; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D; Wang, Jianlong; Wang, Jenny Yingzi

    2014-11-20

    β-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML. © 2014 by The American Society of Hematology.

  20. 3D FDTD modelling of GPR: the effects of antenna polarisation on gpr signals from nonmetal pipes

    International Nuclear Information System (INIS)

    Amiruddin Shaari

    2003-01-01

    A 3D finite-difference time domain (FDTD) modelling of ground penetrating radar (GPR) has been carried out in order to determine the effectiveness of the method when it is used in a ground survey for metal and nonmetal pipes. In particular, the effects of the relative orientation between the antenna polarisation and pipe length and the dielectric contrast between ground soil and pipes on the GPR signal strength are investigated. The results show that the parallel antenna-target is the preferred orientation for metal pipes while the normal or orthogonal arrangement is the preferred one for the nonmetal pipes. The dielectric contrast between medium and target also seems to affect the strength the GPR signals from the nines. (Author)

  1. Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar

    2017-03-27

    Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

  2. Targeting Splicing in Prostate Cancer

    OpenAIRE

    Effrosyni Antonopoulou; Michael Ladomery

    2018-01-01

    Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform speci...

  3. Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

    Science.gov (United States)

    Chang, Junlei; Mancuso, Michael R; Maier, Carolina; Liang, Xibin; Yuki, Kanako; Yang, Lu; Kwong, Jeffrey W; Wang, Jing; Rao, Varsha; Vallon, Mario; Kosinski, Cynthia; Zhang, J J Haijing; Mah, Amanda T; Xu, Lijun; Li, Le; Gholamin, Sharareh; Reyes, Teresa F; Li, Rui; Kuhnert, Frank; Han, Xiaoyuan; Yuan, Jenny; Chiou, Shin-Heng; Brettman, Ari D; Daly, Lauren; Corney, David C; Cheshier, Samuel H; Shortliffe, Linda D; Wu, Xiwei; Snyder, Michael; Chan, Pak; Giffard, Rona G; Chang, Howard Y; Andreasson, Katrin; Kuo, Calvin J

    2017-04-01

    Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

  4. Determination, by using GPR, of the volumetric water content in structures, sub-structures, foundations and soil - ongoing activities in Working Project 2.5 of COST Action TU1208

    Science.gov (United States)

    Tosti, Fabio; Slob, Evert

    2015-04-01

    This work will endeavour to review the current status of research activities carried out in Working Project 2.5 'Determination, by using GPR, of the volumetric water content in structures, sub-structures, foundations and soil' within the framework of Working Group 2 'GPR surveying of pavements, bridges, tunnels and buildings; underground utility and void sensing' of the COST (European COoperation in Science and Technology) Action TU1208 'Civil Engineering Applications of Ground Penetrating Radar' (www.GPRadar.eu). Overall, the Project includes 55 Participants from over 21 countries representing 33 Institutions. By considering the type of Institution, a percentage of 64% (35 units) comes from the academic world, while Research Centres and Companies include, respectively, the 27% (15 units) and 9% (5 units) of Institutions. Geographically speaking, Europe is the continent most represented with 18 out of 21 countries, followed by Africa (2 countries) and Asia (1 country). In more details and according to the Europe sub-regions classification provided by the United Nations, Southern Europe includes 39% of countries, Western Europe 27%, while Northern and Eastern Europe are equally present with 17% of countries each. Relying on the main purpose of Working Project 2.5, namely, the ground-penetrating radar-based evaluation of volumetric water content in structures, substructures , foundations, and soils, four main issues have been overall addressed over the first two years of activities. The first one, has been related to provide a comprehensive state of the art on the topic, due to the wide-ranging applications covered in the main disciplines of civil engineering, differently demanding. In this regard, two main publications reviewing the state of the art have been produced [1,2]. Secondly, discussions among Working Group Chairs and other Working Project Leaders have been undertaken and encouraged to avoid the risk of overlapping amongst similar topics from other Working

  5. Application of GPR for delineating the neotectonic setting and shallow subsurface nature of the seismically active Gedi fault, Kachchh, western India

    International Nuclear Information System (INIS)

    Maurya, D M; Chouksey, V; Joshi, Parul N; Chamyal, L S

    2013-01-01

    The present field and GPR based investigations were carried out along the E–W trending Gedi Fault to precisely constrain the field location and shallow subsurface nature of the fault. The field investigations revealed the presence of thin Quaternary sedimentary cover, especially in the central and western part. Field examination of the scanty exposures showed that the fault trace marks the lithotectonic contact between Mesozoic rocks in the north and the Tertiary (Miocene) sediments to the south. Five sites were selected after field studies for GPR investigations of the Gedi Fault. The well-compacted Mesozoic rocks showed high amplitude returns while the softer and finer grained Tertiary sediments yielded low amplitude returns. The Quaternary sediments are reflected as consistent with wavy reflections in the upper parts of the profiles. The GPR data indicate that the Gedi Fault is a steep north dipping reverse fault which becomes vertical at depth. Since the fault does not displace the Quaternary deposits, we infer that the Gedi Fault has been characterized by low to moderate seismic activity under a compressive stress regime during the late Quaternary period. (paper)

  6. MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2

    Science.gov (United States)

    Coady, Michael J.; El Tarazi, Abdulah; Santer, René; Bissonnette, Pierre; Sasseville, Louis J.; Calado, Joaquim; Lussier, Yoann; Dumayne, Christopher; Bichet, Daniel G.

    2017-01-01

    The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17–SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters. PMID:27288013

  7. The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

    Science.gov (United States)

    Cohen, L; Sekler, I; Hershfinkel, M

    2014-06-26

    The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn(2+) are not well understood. Here, we determined a role of the colonocytic Zn(2+) sensing receptor, ZnR/GPR39, in mediating Zn(2+)-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn(2+)-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn(2+) by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting

  8. Short-chain fatty acids and ketones directly regulate sympathetic nervous system via G protein-coupled receptor 41 (GPR41).

    Science.gov (United States)

    Kimura, Ikuo; Inoue, Daisuke; Maeda, Takeshi; Hara, Takafumi; Ichimura, Atsuhiko; Miyauchi, Satoshi; Kobayashi, Makio; Hirasawa, Akira; Tsujimoto, Gozoh

    2011-05-10

    The maintenance of energy homeostasis is essential for life, and its dysregulation leads to a variety of metabolic disorders. Under a fed condition, mammals use glucose as the main metabolic fuel, and short-chain fatty acids (SCFAs) produced by the colonic bacterial fermentation of dietary fiber also contribute a significant proportion of daily energy requirement. Under ketogenic conditions such as starvation and diabetes, ketone bodies produced in the liver from fatty acids are used as the main energy sources. To balance energy intake, dietary excess and starvation trigger an increase or a decrease in energy expenditure, respectively, by regulating the activity of the sympathetic nervous system (SNS). The regulation of metabolic homeostasis by glucose is well recognized; however, the roles of SCFAs and ketone bodies in maintaining energy balance remain unclear. Here, we show that SCFAs and ketone bodies directly regulate SNS activity via GPR41, a Gi/o protein-coupled receptor for SCFAs, at the level of the sympathetic ganglion. GPR41 was most abundantly expressed in sympathetic ganglia in mouse and humans. SCFA propionate promoted sympathetic outflow via GPR41. On the other hand, a ketone body, β-hydroxybutyrate, produced during starvation or diabetes, suppressed SNS activity by antagonizing GPR41. Pharmacological and siRNA experiments indicated that GPR41-mediated activation of sympathetic neurons involves Gβγ-PLCβ-MAPK signaling. Sympathetic regulation by SCFAs and ketone bodies correlated well with their respective effects on energy consumption. These findings establish that SCFAs and ketone bodies directly regulate GPR41-mediated SNS activity and thereby control body energy expenditure in maintaining metabolic homeostasis.

  9. Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni.

    Science.gov (United States)

    Boroni, Mariana; Sammeth, Michael; Gava, Sandra Grossi; Jorge, Natasha Andressa Nogueira; Macedo, Andréa Mara; Machado, Carlos Renato; Mourão, Marina Moraes; Franco, Glória Regina

    2018-03-01

    Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.

  10. The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer.

    Directory of Open Access Journals (Sweden)

    Zhibin Jing

    Full Text Available Squamous cell carcinoma of the head and neck (SCCHN is an aggressive disease with poor survival and is the sixth most common cancer worldwide. Gastroesophageal reflux is a common event in SCCHN patients. GPR4 is a proton-sensing G-protein coupled receptor, which can be activated by acidosis. The objective of this study was to explore the role of GPR4 in acid exposure and tumor angiogenesis in SCCHN. In this study, we confirmed that overexpressing GPR4 in SCCHN cells could increase the expression and secretion of IL6, IL8 and VEGFA at pH 5.9. This effect could be inhibited by SB203580 (a p38 inhibitor. Western blot analysis indicated that phosphorylation of p38 increased in GPR4 infected cells at pH 5.9, which could be inhibited by SB203580. In tube formation assay, HMEC-1 cells were incubated with conditioned medium (CM, pH 5.9, 6.5, 7.4 derived from control and GPR4 infected SCCHN cells. Tube length was significantly increased in HMEC-1 cells incubated with CM from GPR4 infected cells compared with control cells at pH5.9, which indicated the pro-angiogenic effect of GPR4 in acidic pH. The neutralizing antibodies of IL6, IL8 and VEGFA could inhibit tube formation of HMEC-1 cells. In vivo, the effect of GPR4 on angiogenesis was investigated with the chick chorioallantoic membrane (CAM model. Control and GPR4 infected SCCHN cells were seeded onto the upper CAM surface (n = 5 in each group and 5 μL DMEM/F12 (pH 5.9, 6.5, 7.4 was added to the surface of the cell every 24 h. Four days later, the upper CAM were harvested and the ratio of the vascular area to the CAM area was quantified using Image-Pro Plus 6.0 software. GPR4 infected cells could recruit more vascular than control cells at pH5.9. In conclusion, we suggested that GPR4 induces angiogenesis via GPR4-induced p38-mediated IL6, IL8 and VEGFA secretion at acidic extracellular pH in SCCHN.

  11. Inertial and GPS data integration for positioning and tracking of GPR

    Science.gov (United States)

    Chicarella, Simone; D'Alvano, Alessandro; Ferrara, Vincenzo; Frezza, Fabrizio; Pajewski, Lara

    2015-04-01

    Nowadays many applications and studies use a Global Positioning System (GPS) to integrate Ground-Penetrating Radar (GPR) data [1-2]. The aim is the production of detailed detection maps that are geo-referenced and superimposable on geographic maps themes. GPS provides data to determine static positioning, and to track the mobile detection system path on the land. A low-cost standard GPS, like GPS-622R by RF Solutions Ltd, allows accuracy around 2.5 m CEP (Circular Error Probability), and a maximum update rate of 10 Hz. These accuracy and update rate are satisfying values when we evaluate positioning datum, but they are unsuitable for precision tracking of a speedy-mobile GPR system. In order to determine the relative displacements with respect to an initial position on the territory, an Inertial Measurement Unit (IMU) can be used. Some inertial-system applications for GPR tracking have been presented in recent studies [3-4]. The integration of both GPS and IMU systems is the aim of our work, in order to increase GPR applicability, e.g. the case of a GPR mounted on an unmanned aerial vehicle for the detection of people buried under avalanches [5]. In this work, we will present the design, realization and experimental characterization of our electronic board that includes GPS-622R and AltIMU-10 v3 by Pololu. The latter comprises an inertial-measurement unit and an altimeter. In particular, the IMU adopts L3GD20 gyro and LSM303D accelerometer and magnetometer; the digital barometer LPS331AP provides data for altitude evaluation. The prototype of our system for GPR positioning and tracking is based on an Arduino microcontroller board. Acknowledgement This work benefited from networking activities carried out within the EU funded COST Action TU1208 'Civil Engineering Applications of Ground Penetrating Radar. ' References [1] M. Solla, X. Núñez-Nieto, M. Varela-González, J. Martínez-Sánchez, and P. Arias, 'GPR for Road Inspection: georeferencing and efficient

  12. Investigation of HMA compactability using GPR technique

    Science.gov (United States)

    Plati, Christina; Georgiou, Panos; Loizos, Andreas

    2014-05-01

    . Actually, the prediction is not regulated by any standards or specifications, although the practice is considered to be workable. In view of the above, an extensive experiment was carried out in both the laboratory and the field based on a trial asphalt pavement section under construction. In the laboratory, the study focused on the estimation of the density of HMA specimens achieved through three different roller compaction modes (static, vibratory and a combination of both) targeted to simulate field compaction and assess the asphalt mix compactability. In the field, the different compaction modes were successively implemented on three subsections of the trial pavement section. Along each subsection, GPR data was collected in order to determine the new material's dielectric properties and based on that, to predict its density using proper algorithm. Thus, cores were extracted to be used as ground truth data. The comparison of the new asphalt material compactability as obtained from the laboratory specimens, the predictions based on GPR data and the field cores provided useful information that facilitated the selection of the most effective compaction mode yielding the proper compaction degree in the field. This work benefited from networking activities carried out within the EU funded COST Action TU1208 "Civil Engineering Applications of Ground Penetrating Radar."

  13. Linoleic Acid Permeabilizes Gastric Epithelial Cells by Increasing Connexin43 Levels in the Cell Membrane Via a GPR40- and Akt-Dependent Mechanism

    Science.gov (United States)

    Puebla, Carlos; Cisterna, Bruno A.; Salas, Daniela P.; Delgado-López, Fernando; Lampe, Paul D.; Sáez, Juan C.

    2016-01-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintain the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt. PMID:26869446

  14. Waveform Analysis of UWB GPR Antennas

    Directory of Open Access Journals (Sweden)

    Julia Armesto

    2009-03-01

    Full Text Available Ground Penetrating Radar (GPR systems fall into the category of ultra-wideband (UWB devices. Most GPR equipment covers a frequency range between an octave and a decade by using short-time pulses. Each signal recorded by a GPR gathers a temporal log of attenuated and distorted versions of these pulses (due to the effect of the propagation medium plus possible electromagnetic interferences and noise. In order to make a good interpretation of this data and extract the most possible information during processing, a deep knowledge of the wavelet emitted by the antennas is essential. Moreover, some advanced processing techniques require specific knowledge of this signal to obtain satisfactory results. In this work, we carried out a series of tests in order to determine the source wavelet emitted by a ground-coupled antenna with a 500 MHz central frequency.

  15. Connecting the dots: chromatin and alternative splicing in EMT.

    Science.gov (United States)

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  16. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

  17. Study of a rehabilitated road using GPR and FWD

    Science.gov (United States)

    Marecos, Vania; Fontul, Simona; de Lurdes Antunes, Maria; Solla, Mercedes; Pajewski, Lara

    2017-04-01

    This work focus on the structural evaluation of a rehabilitated road after the conclusion of the first phase of the improvement works. The activities developed in the study comprised the characterization of the pavement layers condition (before the application of the asphalt surface layer) and the prediction of the pavement bearing capacity (taking into account the contribution of the wearing course, to be placed in accordance with the project specifications). For this study two non-destructive tests (NDT) were combined: Falling Weight Deflectometer (FWD) and Ground Penetrating Radar (GPR). The original pavement was essentially composed by a granular layer treated with a bituminous emulsion. The main objectives of the rehabilitation works were the enlargement of the road platform in selected locations, with the construction of a new pavement, and also the reinforcement of the existing pavement to increase its bearing capacity. The FWD tests were performed to assess the bearing capacity of the pavement and were conducted along the outer wheel path, in both directions. The spacing between measurement points was 75 m and the applied impulse load was 50 kN. The results showed a great variability of the deflections measured along the section under study. A preliminary zonation of the pavement was carried out, and was latter adjusted based on the results of the GPR. To determine the thickness of the pavement layers a GPR survey was carried out using a 1.8 GHz antenna and a radar control unit SIR-20, both from GSSI. The GPR tests were performed continuously along the same line as the FWD tests. The GPR tests allowed for the identification of the different structures of the pavement, corresponding to the zones with the new pavement and the existing pavement with reinforcement. Some cores were extracted to calibrate the thickness of the GPR bituminous layers, to verify the conditions of adhesion between layers and also to perform laboratory tests to characterize the

  18. Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?

    Directory of Open Access Journals (Sweden)

    Sushmita Poddar

    2014-06-01

    Full Text Available To date the Simian Virus 40 (SV40 is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

  19. Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

    Science.gov (United States)

    Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

    2017-07-21

    Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for

  20. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  1. Association analyses suggest GPR24 as a shared susceptibility gene for bipolar affective disorder and schizophrenia

    DEFF Research Database (Denmark)

    Severinsen, J E; Als, T D; Binderup, H

    2006-01-01

    (-5) and 0.0006 in the combined group of cases from the Faeroe Islands and Scotland, respectively. The G protein-coupled receptor 24 encoded by GPR24 binds melanin-concentrating hormone (MCH) and has been implicated with feeding behavior, energy metabolism, and regulation of stress and mood. To our knowledge...... comprising 28 distantly related cases (17 BPD, 11 SZ subjects) and 44 controls, and a Scottish sample including 162 patients with BPD, 103 with SZ, and 200 controls. In both samples significant associations were observed in both disorders with predominantly GPR24 SNPs and haplotypes. In the Faeroese sample...

  2. GPR39 signaling is stimulated by zinc ions but not by obestatin

    DEFF Research Database (Denmark)

    Holst, Birgitte; Egerod, Kristoffer L; Schild, Enrico

    2006-01-01

    GPR39 is an orphan member of the ghrelin receptor family that recently was suggested to be the receptor for obestatin, a peptide derived from the ghrelin precursor. Here, we compare the effect of obestatin to the effect of Zn(2+) on signal transduction and study the effect of obestatin on food...... intake. Although Zn(2+) stimulated inositol phosphate turnover, cAMP production, arrestin mobilization, as well as cAMP response element-dependent and serum response element-dependent transcriptional activity in GPR39-expressing cells as opposed to mock-transfected cells, no reproducible effect...... organs such as duodenum and kidney but not in the pituitary and hypothalamus, i.e. presumed central target organs for obestatin. Obestatin had no significant and reproducible effect on acute food intake in either freely fed or fasted lean mice. It is concluded that GPR39 is probably not the obestatin...

  3. The role of the obestatin/GPR39 system in human gastric adenocarcinomas.

    Science.gov (United States)

    Alén, Begoña O; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S; Beiras, Andrés; Casanueva, Felipe F; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P; Pazos, Yolanda

    2016-02-02

    Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

  4. Gigantism: X-linked acrogigantism and GPR101 mutations.

    Science.gov (United States)

    Iacovazzo, Donato; Korbonits, Márta

    X-linked acrogigantism (XLAG) is a recently identified condition of early-onset GH excess resulting from the germline or somatic duplication of the GPR101 gene on chromosome Xq26.3. Thirty patients have been formally reported so far. The disease affects mostly females, occurs usually sporadically, and is characterised by early onset and marked overgrowth. Most patients present with concomitant hyperprolactinaemia. Histopathology shows pituitary hyperplasia or pituitary adenoma with or without associated hyperplasia. XLAG-related pituitary adenomas present peculiar histopathological features that should contribute to raise the suspicion of this rare condition. Treatment is frequently challenging and multi-modal. While females present with germline mutations, the sporadic male patients reported so far were somatic mosaics with variable levels of mosaicism, although no differences in the clinical phenotype were observed between patients with germline or somatic duplication. The GPR101 gene encodes an orphan G protein-coupled receptor normally expressed in the central nervous system, and at particularly high levels in the hypothalamus. While the physiological function and the endogenous ligand of GPR101 are unknown, the high expression of GPR101 in the arcuate nucleus and the occurrence of increased circulating GHRH levels in some patients with XLAG, suggest that increased hypothalamic GHRH secretion could play a role in the pathogenesis of this condition. In this review, we summarise the published evidence on XLAG and GPR101 and discuss the results of recent studies that have investigated the potential role of GPR101 variants in the pathogenesis of pituitary adenomas. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. The emerging role of alternative splicing in senescence and aging.

    Science.gov (United States)

    Deschênes, Mathieu; Chabot, Benoit

    2017-10-01

    Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  6. Online Dictionary Learning Aided Target Recognition In Cognitive GPR

    OpenAIRE

    Giovanneschi, Fabio; Mishra, Kumar Vijay; Gonzalez-Huici, Maria Antonia; Eldar, Yonina C.; Ender, Joachim H. G.

    2017-01-01

    Sparse decomposition of ground penetration radar (GPR) signals facilitates the use of compressed sensing techniques for faster data acquisition and enhanced feature extraction for target classification. In this paper, we investigate the application of an online dictionary learning (ODL) technique in the context of GPR to bring down the learning time as well as improve identification of abandoned anti-personnel landmines. Our experimental results using real data from an L-band GPR for PMN/PMA2...

  7. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin

    2014-01-01

    RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing...

  8. Diacylglycerol kinase α mediates 17-β-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30.

    Science.gov (United States)

    Filigheddu, Nicoletta; Sampietro, Sara; Chianale, Federica; Porporato, Paolo E; Gaggianesi, Miriam; Gregnanin, Ilaria; Rainero, Elena; Ferrara, Michele; Perego, Beatrice; Riboni, Francesca; Baldanzi, Gianluca; Graziani, Andrea; Surico, Nicola

    2011-12-01

    Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-β-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Marta Moreno

    2017-11-01

    Full Text Available A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM.

  10. Electromagnetic modelling, inversion and data-processing techniques for GPR: ongoing activities in Working Group 3 of COST Action TU1208

    Science.gov (United States)

    Pajewski, Lara; Giannopoulos, Antonis; van der Kruk, Jan

    2015-04-01

    This work aims at presenting the ongoing research activities carried out in Working Group 3 (WG3) 'EM methods for near-field scattering problems by buried structures; data processing techniques' of the COST (European COoperation in Science and Technology) Action TU1208 'Civil Engineering Applications of Ground Penetrating Radar' (www.GPRadar.eu). The principal goal of the COST Action TU1208 is to exchange and increase scientific-technical knowledge and experience of GPR techniques in civil engineering, simultaneously promoting throughout Europe the effective use of this safe and non-destructive technique in the monitoring of infrastructures and structures. WG3 is structured in four Projects. Project 3.1 deals with 'Electromagnetic modelling for GPR applications.' Project 3.2 is concerned with 'Inversion and imaging techniques for GPR applications.' The topic of Project 3.3 is the 'Development of intrinsic models for describing near-field antenna effects, including antenna-medium coupling, for improved radar data processing using full-wave inversion.' Project 3.4 focuses on 'Advanced GPR data-processing algorithms.' Electromagnetic modeling tools that are being developed and improved include the Finite-Difference Time-Domain (FDTD) technique and the spectral domain Cylindrical-Wave Approach (CWA). One of the well-known freeware and versatile FDTD simulators is GprMax that enables an improved realistic representation of the soil/material hosting the sought structures and of the GPR antennas. Here, input/output tools are being developed to ease the definition of scenarios and the visualisation of numerical results. The CWA expresses the field scattered by subsurface two-dimensional targets with arbitrary cross-section as a sum of cylindrical waves. In this way, the interaction is taken into account of multiple scattered fields within the medium hosting the sought targets. Recently, the method has been extended to deal with through-the-wall scenarios. One of the

  11. Design of a low cost miniaturized SFCW GPR with initial results

    Science.gov (United States)

    Duggal, Swati; Sinha, Piyush; Gupta, Manish; Patel, Anand; Vedam, V. V.; Mevada, Pratik; Chavda, Rajesh; Shah, Amita; Putrevu, Deepak

    2016-05-01

    This paper discusses about the design &developmental of Ground Penetrating Radar (GPR), various scientific and commercial applications of GPR along with the testing and results of GPR at Antarctica for Ice thickness measurement. GPR instruments are categorised as per their frequency of operation, which is inversely proportional to the depth of penetration. GPRs are also categorized as per method of operation which is time-domain or frequency-domain. Indian market is presently procuring GPRs from only foreign suppliers. Space Applications Centre (SAC) had taken up GPR as R&D Technological development with a view to benchmark the technology which may be transferred to local industry for mass production of instrument at a relatively cheaper cost (~20 times cheaper). Hence, this instrument presents a viable indigenous alternative. Also, the design and configuration was targeted for terrestrial as well as future interplanetary (Lander/Rover) missions of ISRO to map subsurface features. The developed GPR has a very large bandwidth (100%, i.e. bandwidth of 500MHz with centre-frequency of 500MHz) and high dynamic range along with the advantage of being highly portable (<10kg). The system was configured as a Stepped-Frequency-Continuous-Wave (SFCW) GPR which is a frequency domain GPR with the aim to increase the detection capabilities with respect to current systems. In order to achieve this goal, innovative electronic equipment have been designed and developed. Three prototypes were developed and two of them have been delivered for Indian Scientific Expedition to Antarctica (ISEA) in 2013 and 2014-15, respectively and promising results have been obtained. The results from the same closely compare with that from commercial GPR too.

  12. Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition.

    Science.gov (United States)

    Widmayer, Patricia; Kusumakshi, Soumya; Hägele, Franziska A; Boehm, Ulrich; Breer, Heinz

    2017-01-01

    During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice.

  13. The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

    DEFF Research Database (Denmark)

    Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

    2015-01-01

    Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...

  14. Improvement of the energetic properties of the GPR

    Science.gov (United States)

    Pochanin, Gennadiy P.; Ruban, Vadim P.; Kholod, Pavlo V.; Shuba, Alexander A.; Pochanin, Alexander G.; Orlenko, Alexander A.

    2014-05-01

    increase the duration of the samples with the distance. In this way, a smoothing of the noise and an increase of the recorded energy at each subsequent sampling were achieved. The next opportunity to improve the signal to noise ratio is the coherent accumulation of the signal that can be carried out both in digital and analog forms. Due to the fast ADC, it became possible to accumulate a large number of signals in an acceptable survey period. In practice, the amount of accumulated signals is limited by jitter. Thus, to achieve accumulation and re- ception of signals without distortion the authors have suggested and implemented GPR improvements allowing to get the instability of sampling below 3.5 ps. Owing to increase of the pulse-repetition frequency up to 1 MHz and data transmission via Ethernet, it was also possible to provide a fast GPR survey. ----------------------------------------------------------------------------------------- This research has been performed partly owing to EU 7th Framework Marie Curie Actions IRSES project (PIRSES-GA-2010-269157) "Active and Passive Microwaves for Security and Subsurface imaging (AMISS)." The Authors thank COST Action TU1208 "Civil Engineering Applications of Ground Penetrating Radar" for its networking activities.

  15. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray™ design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

  16. Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

    Science.gov (United States)

    Pillaiyar, Thanigaimalai; Köse, Meryem; Sylvester, Katharina; Weighardt, Heike; Thimm, Dominik; Borges, Gleice; Förster, Irmgard; von Kügelgen, Ivar; Müller, Christa E

    2017-05-11

    The G i protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC 50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC 50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

  17. OPTIMAL SIGNAL PROCESSING METHODS IN GPR

    Directory of Open Access Journals (Sweden)

    Saeid Karamzadeh

    2014-01-01

    Full Text Available In the past three decades, a lot of various applications of Ground Penetrating Radar (GPR took place in real life. There are important challenges of this radar in civil applications and also in military applications. In this paper, the fundamentals of GPR systems will be covered and three important signal processing methods (Wavelet Transform, Matched Filter and Hilbert Huang will be compared to each other in order to get most accurate information about objects which are in subsurface or behind the wall.

  18. G Protein-Coupled Receptor 87 (GPR87 Promotes Cell Proliferation in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xia Zhang

    2015-10-01

    Full Text Available G protein-coupled receptor 87 (GPR87 is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87 and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3, but not in the mutant p53 cells (HT1376 and J82. As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells.

  19. Spliced RNA of woodchuck hepatitis virus.

    Science.gov (United States)

    Ogston, C W; Razman, D G

    1992-07-01

    Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

  20. Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

    Directory of Open Access Journals (Sweden)

    Marcin Szczot

    2017-12-01

    Full Text Available Summary: Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. : Szczot et al. find that the mechanoreceptor Piezo2 is extensively alternatively spliced, generating multiple distinct isoforms. Their findings indicate that these splice products have specific tissue and cell type expression patterns and exhibit differences in receptor properties. Keywords: Piezo, touch, sensation, ion-channel, splicing

  1. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site

    NARCIS (Netherlands)

    Mueller, Nancy; Berkhout, Ben; Das, Atze T.

    2015-01-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA

  2. High resolution aquifer characterization using crosshole GPR full-waveform tomography

    Science.gov (United States)

    Gueting, N.; Vienken, T.; Klotzsche, A.; Van Der Kruk, J.; Vanderborght, J.; Caers, J.; Vereecken, H.; Englert, A.

    2016-12-01

    Limited knowledge about the spatial distribution of aquifer properties typically constrains our ability to predict subsurface flow and transport. Here, we investigate the value of using high resolution full-waveform inversion of cross-borehole ground penetrating radar (GPR) data for aquifer characterization. By stitching together GPR tomograms from multiple adjacent crosshole planes, we are able to image, with a decimeter scale resolution, the dielectric permittivity and electrical conductivity of an alluvial aquifer along cross-sections of 50 m length and 10 m depth. A logistic regression model is employed to predict the spatial distribution of lithological facies on the basis of the GPR results. Vertical profiles of porosity and hydraulic conductivity from direct-push, flowmeter and grain size data suggest that the GPR predicted facies classification is meaningful with regard to porosity and hydraulic conductivity, even though the distributions of individual facies show some overlap and the absolute hydraulic conductivities from the different methods (direct-push, flowmeter, grain size) differ up to approximately one order of magnitude. Comparison of the GPR predicted facies architecture with tracer test data suggests that the plume splitting observed in a tracer experiment was caused by a hydraulically low-conductive sand layer with a thickness of only a few decimeters. Because this sand layer is identified by GPR full-waveform inversion but not by conventional GPR ray-based inversion we conclude that the improvement in spatial resolution due to full-waveform inversion is crucial to detect small-scale aquifer structures that are highly relevant for solute transport.

  3. Applications of GPR in archaeological prospecting and cultural heritage diagnostics: Research Perspectives in COST Action TU1208

    Science.gov (United States)

    Pajewski, Lara; Benedetto, Andrea; Schettini, Giuseppe; Soldovieri, Francesco

    2013-04-01

    Ground Penetrating Radar (GPR) is a safe, non-destructive and non-invasive imaging technique that can be effectively used for advanced inspection of composite structures and for diagnostics affecting the whole life-cycle of civil engineering works. GPR can also be successfully employed in archaeological prospecting and cultural heritage diagnostics. In many Countries, where the archeological patrimony is an outstanding value (as Egypt, Israel, Greece, Central and South America), GPR is usually employed both as a diagnostic tool for the preventive detection of archeological structures and as the most advanced instrument able to prospect geometry and shape of underground valuable sites. However many uncertainties persist, because of several difficulties and ambiguities due to the complexity of the image processing in heterogeneous environment. It is possible to identify three main areas, in GPR field, that have to be addressed in order to promote the use of this technology in archaeological prospecting and cultural heritage diagnostics. These are: a) increase of the system sensitivity to enable the usability in a wider range of conditions, archeological sites are often located in impervious and critical environments; b) research novel data processing algorithms/analysis tools for the interpretation of GPR results; c) contribute to the development of new standards and guidelines and to training of end users, that will also help to increase the awareness of operators. It is also important to further investigate and promote a combined use of GPR with other non-invasive advanced techniques, typically used in the archeological investigation. In this framework, the COST Action TU1208 "Civil Engineering Applications of Ground Penetrating Radar", proposed by a research team of "Roma Tre" University, Rome, Italy, has been approved in November 2012 and is going to start in April 2013. It is a 4-years ambitious project already involving 17 European Countries (AT, BE, CH, CZ, DE

  4. A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

    2008-01-01

    RNAs, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly....

  5. Analysis of thin fractures with GPR: from theory to practice

    Science.gov (United States)

    Arosio, Diego; Zanzi, Luigi; Longoni, Laura; Papini, Monica

    2017-04-01

    Whenever we perform a GPR survey to investigate a rocky medium, being the ultimate purpose of the survey either to study the stability of a rock slope or to determine the soundness of a quarried rock block, we would like mainly to detect any fracture within the investigated medium and, possibly, to estimate the parameters of the fractures, namely thickness and filling material. In most of the practical cases, rock fracture thicknesses are very small when compared to the wavelength of the electromagnetic radiation generated by the GPR systems. In such cases, fractures are to be considered as thin beds, i.e. two interfaces whose distance is smaller than GPR resolving capability, and the reflected signal is the sum of the electromagnetic reverberation within the bed. According to this, fracture parameters are encoded in the thin bed complex response and in this work we propose a methodology based on deterministic deconvolution to process amplitude and phase information in the frequency domain to estimate fracture parameters. We first present some theoretical aspects related to thin bed response and a sensitivity analysis concerning fracture thickness and filling. Secondly, we deal with GPR datasets collected both during laboratory experiments and in the facilities of quarrying activities. In the lab tests fractures were simulated by placing materials with known electromagnetic parameters and controlled thickness in between two small marble blocks, whereas field GPR surveys were performed on bigger quarried ornamental stone blocks before they were submitted to the cutting process. We show that, with basic pre-processing and the choice of a proper deconvolving signal, results are encouraging although an ambiguity between thickness and filling estimates exists when no a-priori information is available. Results can be improved by performing CMP radar surveys that are able to provide additional information (i.e., variation of thin bed response versus offset) at the expense

  6. Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition

    Directory of Open Access Journals (Sweden)

    Patricia Widmayer

    2017-08-01

    Full Text Available During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4, which are considered to be receptors for medium and long chain fatty acids (LCFAs, respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA, suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs in the developing gastric mucosa of suckling mice.

  7. Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

    Science.gov (United States)

    Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

    2016-04-01

    Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

  8. Vascular, but not luminal, activation of FFAR1 (GPR40) stimulates GLP-1 secretion from isolated perfused rat small intestine

    DEFF Research Database (Denmark)

    Christensen, Louise Wulff; Kuhre, Rune Ehrenreich; Janus, Charlotte

    2015-01-01

    -protein-cou- pled receptor, FFAR1 (previously GPR40), expressed on L cells and activated by long-chain fatty acids (LCFAs) is a potential target. A link between FFAR1 activation and GLP-1 secretion has been demonstrated in cellular models and small-molecule FFAR1 agonists have been developed. In this study, we exam......- ined the effect of FFAR1 activation on GLP-1 secretion using isolated, per- fused small intestines from rats, a physiologically relevant model allowing distinction between direct and indirect effects of FFAR1 activation. The endogenous FFAR1 ligand, linoleic acid (LA), and four synthetic FFAR1 ago...

  9. Baicalein suppresses 17-β-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

    Science.gov (United States)

    Shang, Dandan; Li, Zheng; Zhu, Zhuxia; Chen, Huamei; Zhao, Lujun; Wang, Xudong; Chen, Yan

    2015-04-01

    Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17β-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell‑Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel G protein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR 30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis.

  10. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

    Directory of Open Access Journals (Sweden)

    Stanley Wong

    Full Text Available Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC, by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

  11. Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

    Directory of Open Access Journals (Sweden)

    Geetha Melangath

    Full Text Available Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional

  12. Stable GPR101 over-Expressing Cell Lines As an Invaluable Tool for Functional Studies, Ligand Screening, and the Identification of Deregulated Genes/Pathways in Patients with X-Linked Acrogigantism

    OpenAIRE

    Trivellin, Giampaolo; Janjic, Maria; Larco, Darwin; Tomic, Melanija; Daly, Adrian; Palmeira, Leonor; Faucz; BECKERS, Albert; Wu, T John; Calebiro, Davide; Stojilkovic, Stanko; Stratakis, Constantine

    2017-01-01

    Background: GPR101 is an orphan G protein-coupled receptor (GPCR) that is duplicated in patients with X-linked acrogigantism (X-LAG) and over-expressed in their GH- and PRL-secreting tumors. GPR101 is a constitutively active GPCR that strongly activates the cAMP pathway. To elucidate the mechanisms through which GPR101 causes GH over-secretion we generated HEK293 and GH/PRL-secreting (GH3) cells with stable GPR101 expression. Methods: Both cell lines were created via direct integration of ...

  13. GPR measurements of attenuation in concrete

    Science.gov (United States)

    Eisenmann, David; Margetan, Frank J.; Pavel, Brittney

    2015-03-01

    Ground-penetrating radar (GPR) signals from concrete structures are affected by several phenomenon, including: (1) transmission and reflection coefficients at interfaces; (2) the radiation patterns of the antenna(s) being used; and (3) the material properties of concrete and any embedded objects. In this paper we investigate different schemes for determining the electromagnetic (EM) attenuation of concrete from measured signals obtained using commercially-available GPR equipment. We adapt procedures commonly used in ultrasonic inspections where one compares the relative strengths of two or more signals having different travel paths through the material of interest. After correcting for beam spread (i.e., diffraction), interface phenomena, and equipment amplification settings, any remaining signal differences are assumed to be due to attenuation thus allowing the attenuation coefficient (say, in dB of loss per inch of travel) to be estimated. We begin with a brief overview of our approach, and then discuss how diffraction corrections were determined for our two 1.6 GHz GPR antennas. We then present results of attenuation measurements for two types of concrete using both pulse/echo and pitch/catch measurement setups.

  14. GPR measurements of attenuation in concrete

    International Nuclear Information System (INIS)

    Eisenmann, David; Margetan, Frank J.; Pavel, Brittney

    2015-01-01

    Ground-penetrating radar (GPR) signals from concrete structures are affected by several phenomenon, including: (1) transmission and reflection coefficients at interfaces; (2) the radiation patterns of the antenna(s) being used; and (3) the material properties of concrete and any embedded objects. In this paper we investigate different schemes for determining the electromagnetic (EM) attenuation of concrete from measured signals obtained using commercially-available GPR equipment. We adapt procedures commonly used in ultrasonic inspections where one compares the relative strengths of two or more signals having different travel paths through the material of interest. After correcting for beam spread (i.e., diffraction), interface phenomena, and equipment amplification settings, any remaining signal differences are assumed to be due to attenuation thus allowing the attenuation coefficient (say, in dB of loss per inch of travel) to be estimated. We begin with a brief overview of our approach, and then discuss how diffraction corrections were determined for our two 1.6 GHz GPR antennas. We then present results of attenuation measurements for two types of concrete using both pulse/echo and pitch/catch measurement setups

  15. GPR measurements of attenuation in concrete

    Energy Technology Data Exchange (ETDEWEB)

    Eisenmann, David, E-mail: djeisen@cnde.iastate.edu; Margetan, Frank J., E-mail: djeisen@cnde.iastate.edu; Pavel, Brittney, E-mail: djeisen@cnde.iastate.edu [Center for Nondestructive Evaluation, Iowa State University, 1915 Scholl Road, Ames, IA 50011-3042 (United States)

    2015-03-31

    Ground-penetrating radar (GPR) signals from concrete structures are affected by several phenomenon, including: (1) transmission and reflection coefficients at interfaces; (2) the radiation patterns of the antenna(s) being used; and (3) the material properties of concrete and any embedded objects. In this paper we investigate different schemes for determining the electromagnetic (EM) attenuation of concrete from measured signals obtained using commercially-available GPR equipment. We adapt procedures commonly used in ultrasonic inspections where one compares the relative strengths of two or more signals having different travel paths through the material of interest. After correcting for beam spread (i.e., diffraction), interface phenomena, and equipment amplification settings, any remaining signal differences are assumed to be due to attenuation thus allowing the attenuation coefficient (say, in dB of loss per inch of travel) to be estimated. We begin with a brief overview of our approach, and then discuss how diffraction corrections were determined for our two 1.6 GHz GPR antennas. We then present results of attenuation measurements for two types of concrete using both pulse/echo and pitch/catch measurement setups.

  16. An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

    Science.gov (United States)

    Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

    2017-08-21

    Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

    KAUST Repository

    Nagashima, Yukihiro

    2011-07-01

    IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

  18. Kiss-1/GPR54 protein expression in breast cancer.

    Science.gov (United States)

    Papaoiconomou, Eleni; Lymperi, Maria; Petraki, Constantina; Philippou, Anastassios; Msaouel, Pavlos; Michalopoulou, Fani; Kafiri, Georgia; Vassilakos, George; Zografos, Georgios; Koutsilieris, Michael

    2014-03-01

    Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (pbreast cancer and fibrocystic disease specimens. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

  19. Improvement of vascular function by acute and chronic treatment with the GPR30 agonist G1 in experimental diabetes mellitus.

    Directory of Open Access Journals (Sweden)

    Zi-lin Li

    Full Text Available The G-protein coupled estrogen receptor 30 (GPR30 is a seven-transmembrane domain receptor that mediates rapid estrogen responses in a wide variety of cell types. This receptor is highly expressed in the cardiovascular system, and exerts vasodilatory effects. The objective of the present study was to investigate the effects of GPR30 on vascular responsiveness in diabetic ovariectomized (OVX rats and elucidate the possible mechanism involved. The roles of GPR30 were evaluated in the thoracic aorta and cultured endothelial cells. The GPR30 agonist G1 induced a dose-dependent vasodilation in the thoracic aorta of the diabetic OVX rats, which was partially attenuated by the nitric oxide synthase (NOS inhibitor, nitro-L-arginine methylester (L-NAME and the GPR30-selective antagonist G15. Dose-dependent vasoconstrictive responses to phenylephrine were attenuated significantly in the rings of the thoracic aorta following the acute G1 administration in the diabetic OVX rats. This effect of G1 was abolished partially by L-NAME and G15. The acute administration of G1 increased significantly the eNOS activity and the concentration of NO in the endothelial cells exposed to high glucose. G1 treatment induced an enhanced endothelium-dependent relaxation to acetylcholine (Ach in the diabetic OVX rats. Further examination revealed that G1 induced vasodilation in the diabetic OVX rats by increasing the phosphorylation of eNOS. These findings provide preliminary evidence that GPR30 activation leads to eNOS activation, as well as vasodilation, to a certain degree and has beneficial effects on vascular function in diabetic OVX rats.

  20. GPR applications in Civil Engineering in Spain - state-of-the-art

    Science.gov (United States)

    Pérez Gracia, Vega; Solla, Mercedes; Santos-Assunçao, Sonia; Lorenzo, Henrique

    2014-05-01

    GPR was introduced in Spain in 1990, and the first significant work was the PhD thesis of H. Lorenzo in 1994. Due to its versatile applicability, the employ has been increased and actually, GPR is extensively used in detection of pipes, wiring and urban services mainly. During the last years, this method was also widely utilized in the detection of graves from the civil war and in forensic studies, with irregular results. It was also commonly applied in archaeology. Actually exists more than 20 private companies offering geotechnical services by means of GPR. Also, several public institutions as Universities and Research Institutes base part of their research in GPR or in GPR applications. Notwithstanding, no training courses of specific formation on GPR is offered, but in several doctorate programs it is possible to work with GPR. Also, in many schools, GPR is part of the geophysical formation of graduate students. However, no national guidelines and rules exist, and each company defines the investigation protocols. Nevertheless, one of the aims of the Comisión Española de Geodesia y Geofísica (Spanish Committee for Geodesy and Geophysics) is to define guidelines for the GPR studies. Probably, the existence of national guidelines or perhaps European guidelines could be the most effective way to promote the responsible use of GPR in different domains. On the other hand, perhaps recommendations on the use of combined methodologies could be a practical way to persuade in the application of geophysical non-destructive technologies. The CEDEX, Centro de Estudios y Experimentación de Obras Públicas (Center for Studies and Experimentation in Civil Engineering), which is a civil engineering research agency in Spain, offers different test sites to calibrate and evaluate the method. It is an autonomous organization, organically ascribed at present to the Ministry of Fomento, and functionally ascribed to the Ministries of Fomento and Medioambiente of Spain, giving

  1. Identification of buried victims in natural disaster with GPR method

    Science.gov (United States)

    Dewi, Rianty Kusuma; Kurniawan, Adityo; Taqwantara, Reyhan Fariz; Iskandar, Farras M.; Naufal, Taufiq Ziyan; Widodo

    2017-07-01

    Indonesian is one of the most seismically active regions in the world and has very complicated plate convergence because there is meeting point of several tectonic plates. The complexity of tectonic features causes a lot of natural disasters such as landslides, tsunamis, earth quakes, volcanoes eruption, etc. Sometimes, the disasters occurs in high populated area and causing thousands to millions of victim been buried under the rumble. Unfortunately, the evacuation still uses the conventional method such using rescue dogs whereas the sensitivity of smell is decrease when the victims buried under the level of the ground. The purpose of this study is to detect buried bodies using GPR method, so it can enhance the effectiveness and the efficiency in looking for the disaster victims. GPR method is used because it can investigate things under the ground. A detailed GPR research has been done in Cikutra Graveyard, Bandung, with corpse buried two week until two years before the research. The radar profiles from this research showed amplitude contras anomaly between the new corpse and the old ones. We obtained the amplitude contras at 1.2-1.4 meters under the surface. This method proved to be effective but still need more attention on undulated surface and non-soil areas.

  2. The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.

    Science.gov (United States)

    Venkataraman, Chandrasekar; Kuo, Frederick

    2005-11-15

    The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biological function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-gamma production in GPR84(-/-) mice compared to wild-type mice. Augmented IL-4 production in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-gamma steady-state mRNA in GPR84(-/-) T cells. In addition, Th2 effector cells generated in vitro from GPR84(-/-) mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 production between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.

  3. TOMOGRAPHIC SITE CHARACTERIZATION USING CPT, ERT, AND GPR

    Energy Technology Data Exchange (ETDEWEB)

    Rexford M. Morey; Susanne M. Conklin; Stephen P. Farrington, P.E.; James D. Shinn II, P.E.

    1999-07-01

    The US Department of Energy (DOE) is responsible for the cleanup of inactive DOE sites and for bringing DOE sites and facilities into compliance with federal, state, and local laws and regulations. The DOE's Office of Environmental Management (EM) needs advanced technologies that can make environmental restoration and waste management operations more efficient and less costly. These techniques are required to better characterize the physical, hydrogeological, and chemical properties of the subsurface while minimizing and optimizing the use of boreholes and monitoring wells. Today the cone penetrometer technique (CPT) is demonstrating the value of a minimally invasive deployment system for site characterization. Applied Research Associates, Inc. is developing two new sensor packages for site characterization and monitoring. The two new methods are: (1) Electrical Resistivity Tomography (ERT); and (2) Ground Penetrating Radar (GPR) Tomography. These sensor systems are now integrated with the CPT. The results of this program now make it possible to install ERT and GPR units by CPT methods and thereby reduce installation costs and total costs for ERT and GPR surveys. These two techniques can complement each other in regions of low resistivity where ERT is more effective and regions of high resistivity where GPR is more effective. The results show that CPT-installed GeoWells can be used for both ERT and GPR borehole tomographic subsurface imaging. These two imaging techniques can be used for environmental site characterization and monitoring have numerous and diverse applications within site cleanup and waste management operations.

  4. Male mice that lack the G-protein-coupled receptor GPR41 have low energy expenditure and increased body fat content.

    Science.gov (United States)

    Bellahcene, Mohamed; O'Dowd, Jacqueline F; Wargent, Ed T; Zaibi, Mohamed S; Hislop, David C; Ngala, Robert A; Smith, David M; Cawthorne, Michael A; Stocker, Claire J; Arch, Jonathan R S

    2013-05-28

    SCFA are produced in the gut by bacterial fermentation of undigested carbohydrates. Activation of the Gαi-protein-coupled receptor GPR41 by SCFA in β-cells and sympathetic ganglia inhibits insulin secretion and increases sympathetic outflow, respectively. A possible role in stimulating leptin secretion by adipocytes is disputed. In the present study, we investigated energy balance and glucose homoeostasis in GPR41 knockout mice fed on a standard low-fat or a high-fat diet. When fed on the low-fat diet, body fat mass was raised and glucose tolerance was impaired in male but not female knockout mice compared to wild-type mice. Soleus muscle and heart weights were reduced in the male mice, but total body lean mass was unchanged. When fed on the high-fat diet, body fat mass was raised in male but not female GPR41 knockout mice, but by no more in the males than when they were fed on the low-fat diet. Body lean mass and energy expenditure were reduced in male mice but not in female knockout mice. These results suggest that the absence of GPR41 increases body fat content in male mice. Gut-derived SCFA may raise energy expenditure and help to protect against obesity by activating GPR41.

  5. Fine-scale variation and genetic determinants of alternative splicing across individuals.

    Directory of Open Access Journals (Sweden)

    Jasmin Coulombe-Huntington

    2009-12-01

    Full Text Available Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre-mRNA splicing (AS in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72% candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and

  6. Supplementary Material for: Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar; Ling, Yu; Butt, Haroon; Mariappan, Kiruthiga; Benhamed, Moussa; Mahfouz, Magdy

    2017-01-01

    Abstract Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

  7. Analyses of GPR signals for characterization of ground conditions in urban areas

    Science.gov (United States)

    Hong, Won-Taek; Kang, Seonghun; Lee, Sung Jin; Lee, Jong-Sub

    2018-05-01

    Ground penetrating radar (GPR) is applied for the characterization of the ground conditions in urban areas. In addition, time domain reflectometry (TDR) and dynamic cone penetrometer (DCP) tests are conducted for the accurate analyses of the GPR images. The GPR images are acquired near a ground excavation site, where a ground subsidence occurred and was repaired. Moreover, the relative permittivity and dynamic cone penetration index (DCPI) are profiled through the TDR and DCP tests, respectively. As the ground in the urban area is kept under a low-moisture condition, the relative permittivity, which is inversely related to the electromagnetic impedance, is mainly affected by the dry density and is inversely proportional to the DCPI value. Because the first strong signal in the GPR image is shifted 180° from the emitted signal, the polarity of the electromagnetic wave reflected at the dense layer, where the reflection coefficient is negative, is identical to that of the first strong signal. The temporal-scaled GPR images can be accurately converted into the spatial-scaled GPR images using the relative permittivity determined by the TDR test. The distribution of the loose layer can be accurately estimated by using the spatial-scaled GPR images and reflection characteristics of the electromagnetic wave. Note that the loose layer distribution estimated in this study matches well with the DCPI profile and is visually verified from the endoscopic images. This study demonstrates that the GPR survey complemented by the TDR and DCP tests, may be an effective method for the characterization of ground conditions in an urban area.

  8. Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation.

    Science.gov (United States)

    Emgård, Johanna; Kammoun, Hana; García-Cassani, Bethania; Chesné, Julie; Parigi, Sara M; Jacob, Jean-Marie; Cheng, Hung-Wei; Evren, Elza; Das, Srustidhar; Czarnewski, Paulo; Sleiers, Natalie; Melo-Gonzalez, Felipe; Kvedaraite, Egle; Svensson, Mattias; Scandella, Elke; Hepworth, Matthew R; Huber, Samuel; Ludewig, Burkhard; Peduto, Lucie; Villablanca, Eduardo J; Veiga-Fernandes, Henrique; Pereira, João P; Flavell, Richard A; Willinger, Tim

    2018-01-16

    Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

    Science.gov (United States)

    Bhat-Nakshatri, Poornima; Song, Eun-Kyung; Collins, Nikail R; Uversky, Vladimir N; Dunker, A Keith; O'Malley, Bert W; Geistlinger, Tim R; Carroll, Jason S; Brown, Myles; Nakshatri, Harikrishna

    2013-06-11

    Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ER

  10. Double-Sided Sliding-Paraboloid (DSSP): A new tool for preprocessing GPR data

    Science.gov (United States)

    Rashed, Mohamed; Rashed, Essam A.

    2017-05-01

    Background noise in Ground Penetrating Radar (GPR) data is a nagging problem that degrades the quality of GPR images and increases their ambiguity. There are several methods adopting different strategies to remove background noise. In this study, we present the Double-Sided Sliding-Paraboloid (DSSP) as a new background removal technique. Experiments conducted on field GPR data show that the proposed DSSP technique has several advantages over existing background removal techniques. DSSP removes background noise more efficiently while preserving first arrivals and other strong horizontal reflections. Moreover, DSSP introduces no artifacts to GPR data and corrects data for DC-shift and wow noise.

  11. Position dependence of the rous sarcoma virus negative regulator of splicing element reflects proximity to a 5' splice site

    International Nuclear Information System (INIS)

    Wang Yuedi; McNally, Mark T.

    2003-01-01

    Rous sarcoma virus (RSV) requires incomplete splicing of its viral transcripts to maintain efficient replication. A splicing inhibitor element, the negative regulator of splicing (NRS), is located near the 5' end of the RNA but the significance of this positioning is not known. In a heterologous intron the NRS functions optimally when positioned close to the authentic 5' splice site. This observation led us to investigate the basis of the position dependence. Four explanations were put forth and stressed the role of three major elements involved in splicing, the 3' splice site, the 5' splice site, and the 5' end cap structure. NRS function was unrelated to its position relative to the 3' splice site or the cap structure and appeared to depend on its position relative to the authentic 5' splice site. We conclude that position dependence may reflect distance constraints necessary for competition of the NRS with the authentic 5' splice site for pairing with the 3' splice sites

  12. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    Science.gov (United States)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  13. Molecular mechanism of Zn2+ agonism in the extracellular domain of GPR39

    DEFF Research Database (Denmark)

    Storjohann, Laura; Holst, Birgitte; Schwartz, Thue W

    2008-01-01

    -terminal segment. Surprisingly, substitution of Asp313 located in extracellular loop 3 greatly increased ligand-independent signaling and apparently eliminated Zn2+-induced activation. It is proposed that Zn2+ acts as an agonist for GPR39, not in the classical manner by directly stabilizing an active conformation...

  14. Zinc Supplementation, via GPR39, Upregulates PKCζ to Protect Intestinal Barrier Integrity in Caco-2 Cells Challenged by Salmonella enterica Serovar Typhimurium.

    Science.gov (United States)

    Shao, Yu-Xin; Lei, Zhao; Wolf, Patricia G; Gao, Yan; Guo, Yu-Ming; Zhang, Bing-Kun

    2017-07-01

    Background: Zinc has been shown to improve intestinal barrier function against Salmonella enterica serovar Typhimurium ( S. typhimurium ) infection, but the mechanisms involved in this process remain undefined. Objective: We aimed to explore the roles of G protein-coupled receptor (GPR)39 and protein kinase Cζ (PKCζ) in the regulation by zinc of intestinal barrier function. Methods: A Transwell Caco-2 monolayer was pretreated with 0, 50, or 100 μM Zn and then incubated with S. typhimurium for 0-6 h. Afterward, cells silenced by the small interfering RNA for GPR39 or PKCζ were pretreated with 100 μM Zn and incubated with S. typhimurium for 3 h. Finally, transepithelial electrical resistance (TEER), permeability, tight junction (TJ) proteins, and signaling molecules GPR39 and PKCζ were measured. Results: Compared with controls, S. typhimurium decreased TEER by 62.3-96.2% at 4-6 h ( P 0.1). Silencing GPR39 decreased ( P zinc-activated PKCζ and blocked ( P zinc on epithelial integrity. Furthermore, silencing PKCζ counteracted the protective effect of zinc on epithelial integrity but did not inhibit GPR39 ( P = 0.138). Conclusion: We demonstrated that zinc upregulates PKCζ by activating GPR39 to enhance the abundance of ZO-1, thereby improving epithelial integrity in S. typhimurium- infected Caco-2 cells. © 2017 American Society for Nutrition.

  15. Targeting GPR120 and other fatty acid sensing GPCRs ameliorates insulin resistance and inflammatory diseases

    Science.gov (United States)

    Talukdar, Saswata; Olefsky, Jerrold M; Osborn, Olivia

    2011-01-01

    The last decade has seen great progress in the understanding of the molecular pharmacology, physiological function and therapeutic potential of the G protein-coupled receptors. Free Fatty acids (FFAs) have been demonstrated to act as ligands of several GPCRs including GPR40, GPR43, GPR84, GPR119 and GPR120. We have recently shown that GPR120 acts as a physiological receptor of ω3 fatty acids in macrophages and adipocytes, which mediate potent anti-inflammatory and insulin sensitizing effects. The important role GPR120 plays in the control of inflammation raises the possibility that targeting this receptor could have therapeutic potential in many inflammatory diseases including obesity and type 2 diabetes. In this review, we discuss lipid-sensing GPCRs and highlight potential outcomes of targeting such receptors in ameliorating disease. PMID:21663979

  16. Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84.

    Science.gov (United States)

    Mahmud, Zobaer Al; Jenkins, Laura; Ulven, Trond; Labéguère, Frédéric; Gosmini, Romain; De Vos, Steve; Hudson, Brian D; Tikhonova, Irina G; Milligan, Graeme

    2017-12-20

    Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine 172 , located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [ 3 H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine 172 . These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.

  17. G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

    Science.gov (United States)

    White, James P; Wrann, Christiane D; Rao, Rajesh R; Nair, Sreekumaran K; Jedrychowski, Mark P; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P; Ruas, Jorge L; Hornberger, Troy A; Wu, Zhidan; Glass, David J; Piao, Xianhua; Spiegelman, Bruce M

    2014-11-04

    Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

  18. Surface-based GPR underestimates below-stump root biomass

    Science.gov (United States)

    John R. Butnor; Lisa J. Samuelson; Thomas A. Stokes; Kurt H. Johnsen; Peter H. Anderson; Carlos A. Gonzalez-Benecke

    2016-01-01

    Aims While lateral root mass is readily detectable with ground penetrating radar (GPR), the roots beneath a tree (below-stump) and overlapping lateral roots near large trees are problematic for surface-based antennas operated in reflection mode. We sought to determine if tree size (DBH) effects GPR root detection proximal to longleaf pine (Pinus palustris Mill) and if...

  19. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

    This book is an up-to-date treatment of optical fiber fusion splicing incorporating all the recent innovations in the field. It provides a toolbox of general strategies and specific techniques that the reader can apply when optimizing fusion splices between novel fibers. It specifically addresses considerations important for fusion splicing of contemporary specialty fibers including dispersion compensating fiber, erbium-doped gain fiber, polarization maintaining fiber, and microstructured fiber. Finally, it discusses the future of optical fiber fusion splicing including silica and non-silica based optical fibers as well as the trend toward increasing automation. Whilst serving as a self-contained reference work, abundant citations from the technical literature will enable readers to readily locate primary sources.

  20. Sex determination in insects: a binary decision based on alternative splicing.

    Science.gov (United States)

    Salz, Helen K

    2011-08-01

    The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex-determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein-encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein-encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. The neurogenetics of alternative splicing.

    Science.gov (United States)

    Vuong, Celine K; Black, Douglas L; Zheng, Sika

    2016-05-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.

  2. LRRTM3 Regulates Excitatory Synapse Development through Alternative Splicing and Neurexin Binding

    Directory of Open Access Journals (Sweden)

    Ji Won Um

    2016-02-01

    Full Text Available The four members of the LRRTM family (LRRTM1-4 are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.

  3. Novel G Protein-Coupled Oestrogen Receptor GPR30 Shows Changes in mRNA Expression in the Rat Brain over the Oestrous Cycle

    Directory of Open Access Journals (Sweden)

    Emma J. Spary

    2012-02-01

    Full Text Available Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and β in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS, ventrolateral medulla (VLM and periaqueductal gray (PAG GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

  4. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    Directory of Open Access Journals (Sweden)

    Penny David

    2007-10-01

    Full Text Available Abstract Background Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. Results For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants show high levels of alternative splicing. Genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. Conclusion Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process.

  5. Targeting GPR120 and other fatty acid-sensing GPCRs ameliorates insulin resistance and inflammatory diseases.

    Science.gov (United States)

    Talukdar, Saswata; Olefsky, Jerrold M; Osborn, Olivia

    2011-09-01

    The past decade has seen great progress in the understanding of the molecular pharmacology, physiological function and therapeutic potential of G-protein-coupled receptors (GPCRs). Free fatty acids (FFAs) have been demonstrated to act as ligands of several GPCRs including GPR40, GPR43, GPR84, GPR119 and GPR120. We have recently shown that GPR120 acts as a physiological receptor of ω3 fatty acids in macrophages and adipocytes, which mediate potent anti-inflammatory and insulin sensitizing effects. The important role GPR120 plays in the control of inflammation raises the possibility that targeting this receptor could have therapeutic potential in many inflammatory diseases including obesity and type 2 diabetes. In this review paper, we discuss lipid-sensing GPCRs and highlight potential outcomes of targeting such receptors in ameliorating disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

    Science.gov (United States)

    Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

    2017-10-30

    Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

  7. BAP1 missense mutation c.2054 A>T (p.E685V completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

    Full Text Available BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM, clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val, identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1 a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU, which resulted in the deletion of 4 base pairs and presumably protein truncation; 2 a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3 partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing.

  8. FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

    Science.gov (United States)

    Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

    2012-01-01

    Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

  9. SpliceSeq: a resource for analysis and visualization of RNA-Seq data on alternative splicing and its functional impacts.

    Science.gov (United States)

    Ryan, Michael C; Cleland, James; Kim, RyangGuk; Wong, Wing Chung; Weinstein, John N

    2012-09-15

    SpliceSeq is a resource for RNA-Seq data that provides a clear view of alternative splicing and identifies potential functional changes that result from splice variation. It displays intuitive visualizations and prioritized lists of results that highlight splicing events and their biological consequences. SpliceSeq unambiguously aligns reads to gene splice graphs, facilitating accurate analysis of large, complex transcript variants that cannot be adequately represented in other formats. SpliceSeq is freely available at http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview. The application is a Java program that can be launched via a browser or installed locally. Local installation requires MySQL and Bowtie. mryan@insilico.us.com Supplementary data are available at Bioinformatics online.

  10. Simulation for ground penetrating radar (GPR) study of the subsurface structure of the Moon

    Science.gov (United States)

    Fa, Wenzhe

    2013-12-01

    Ground penetrating radar (GPR) is currently within the scope of China's Chang-E 3 lunar mission, to study the shallow subsurface of the Moon. In this study, key factors that could affect a lunar GPR performance, such as frequency, range resolution, and antenna directivity, are discussed firstly. Geometrical optics and ray tracing techniques are used to model GPR echoes, considering the transmission, attenuation, reflection, geometrical spreading of radar waves, and the antenna directivity. The influence on A-scope GPR echoes and on the simulated radargrams for the Sinus Iridum region by surface and subsurface roughness, dielectric loss of the lunar regolith, radar frequency and bandwidth, and the distance between the transmit and receive antennas are discussed. Finally, potential scientific return about lunar subsurface properties from GPR echoes is also discussed. Simulation results suggest that subsurface structure from several to hundreds of meters can be studied from GPR echoes at P and VHF bands, and information about dielectric permittivity and thickness of subsurface layers can be estimated from GPR echoes in combination with regolith composition data.

  11. Alternative REST Splicing Underappreciated

    OpenAIRE

    Chen, Guo-Lin; Miller, Gregory

    2017-01-01

    As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription, an N-terminal REST4 isoform caused by alternative splicing - inclusion of an extra exon (N3c) which introduces a pre-mature stop codon - has been implicated in...

  12. ß-Cell Specific Overexpression of GPR39 Protects against Streptozotocin-Induced Hyperglycemia

    DEFF Research Database (Denmark)

    Egerod, Kristoffer Lihme; Jin, Chunyu; Petersen, Pia Steen

    2011-01-01

    and OGTT. Although the overexpression of the constitutively active GPR39 receptor in animals not treated with streptozotocin appeared by itself to impair the glucose tolerance slightly and to decrease the ß-cell mass, it nevertheless totally protected against the gradual hyperglycemia in the steptozotocin...

  13. GPR39 Zn2+-sensing receptor

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Singewald, Nicolas; Holst, Birgitte

    2015-01-01

    ). Preclinical studies provide evidence that zinc deficiency leads to depressive-like behavior related to down-regulation of the GPR39 Zn2+-sensing receptor. Zinc binds to the GPR39 and triggers signals, leading to CRE-dependent gene transcription, resulting in increases in proteins such as brain...

  14. Alternative RNA splicing and cancer

    Science.gov (United States)

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  15. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  16. GPR39 is coupled to TMEM16A in intestinal fibroblast-like cells.

    Directory of Open Access Journals (Sweden)

    Fanning Zeng

    Full Text Available GPR39 is a GPCR implicated as a regulator of gastrointestinal motility, although the mechanism remains elusive. Here, we report that GPR39 is expressed by a specific cell population cultured from mouse small intestine muscle layers, which was subsequently identified as fibroblast-like cells (FLCs that have recently been shown to modulate gut motility. Application of the GPR39 agonist, Zn(2+, induced large currents and membrane depolarization in FLCs cultured from wild-type mice, but not Gpr39(-/- mice. This Zn(2+-induced current could be suppressed by application of a TMEM16A antagonist, CaCC(inh-A01, or by silencing Tmem16a expression. These data suggest that GPR39 might modulate gut motility via regulating TMEM16A function in FLCs.

  17. Aberrant alternative splicing is another hallmark of cancer.

    Science.gov (United States)

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  18. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    OpenAIRE

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  19. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    International Nuclear Information System (INIS)

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-01-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten -/- fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten -/- cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten -/- cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  20. GPR survey, as one of the best geophysical methods for social and industrial needs

    Science.gov (United States)

    Chernov, Anatolii

    2016-04-01

    activity. Monitoring of such hazards as landslides, underground erosion, variation in ground water level can help prevent dangerous processes with destructive consequences, which can result in peoples' injuries and even death. Moreover, GPR can be used in other spheres of life, where investigation of hidden (under or behind conductive for electromagnetic wave material) objects is needed: rescue operations (finding of people after natural and human-made disasters under snow, under debris of building material); military purpose (security systems, identification of people presence through walls, doors, ground etc.). Author work on algorithms (first of all for VIY GPRs (http://viy.ua/)), which will help more precisely find objects of interest on radarograms and even solve inverse problem of geophysics. According to information in that article, geophysical methods can be widely used to solve great variety of tasks and help to investigate humans' past (researches of cultural heritage) and provide information to create safe and comfortable future (preventing of natural hazards and better planning of construction).

  1. A novel CDX2 isoform regulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Matthew E Witek

    Full Text Available Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain. CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2 and pre-mRNA processing (CDX2/AS.

  2. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    Science.gov (United States)

    Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

    2012-01-01

    Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

  3. Analysis of a FANCE Splice Isoform in Regard to DNA Repair.

    Science.gov (United States)

    Bouffard, Frédérick; Plourde, Karine; Bélanger, Simon; Ouellette, Geneviève; Labrie, Yvan; Durocher, Francine

    2015-09-25

    The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. GPR Laboratory Tests For Railways Materials Dielectric Properties Assessment

    Directory of Open Access Journals (Sweden)

    Francesca De Chiara

    2014-10-01

    Full Text Available In railways Ground Penetrating Radar (GPR studies, the evaluation of materials dielectric properties is critical as they are sensitive to water content, to petrographic type of aggregates and to fouling condition of the ballast. Under the load traffic, maintenance actions and climatic effects, ballast condition change due to aggregate breakdown and to subgrade soils pumping, mainly on existing lines with no sub ballast layer. The main purpose of this study was to validate, under controlled conditions, the dielectric values of materials used in Portuguese railways, in order to improve the GPR interpretation using commercial software and consequently the management maintenance planning. Different materials were tested and a broad range of in situ conditions were simulated in laboratory, in physical models. GPR tests were performed with five antennas with frequencies between 400 and 1800 MHz. The variation of the dielectric properties was measured, and the range of values that can be obtained for different material condition was defined. Additionally, in situ GPR measurements and test pits were performed for validation of the dielectric constant of clean ballast. The results obtained are analyzed and the main conclusions are presented herein.

  5. IMF-Slices for GPR Data Processing Using Variational Mode Decomposition Method

    Directory of Open Access Journals (Sweden)

    Xuebing Zhang

    2018-03-01

    Full Text Available Using traditional time-frequency analysis methods, it is possible to delineate the time-frequency structures of ground-penetrating radar (GPR data. A series of applications based on time-frequency analysis were proposed for the GPR data processing and imaging. With respect to signal processing, GPR data are typically non-stationary, which limits the applications of these methods moving forward. Empirical mode decomposition (EMD provides alternative solutions with a fresh perspective. With EMD, GPR data are decomposed into a set of sub-components, i.e., the intrinsic mode functions (IMFs. However, the mode-mixing effect may also bring some negatives. To utilize the IMFs’ benefits, and avoid the negatives of the EMD, we introduce a new decomposition scheme termed variational mode decomposition (VMD for GPR data processing for imaging. Based on the decomposition results of the VMD, we propose a new method which we refer as “the IMF-slice”. In the proposed method, the IMFs are generated by the VMD trace by trace, and then each IMF is sorted and recorded into different profiles (i.e., the IMF-slices according to its center frequency. Using IMF-slices, the GPR data can be divided into several IMF-slices, each of which delineates a main vibration mode, and some subsurface layers and geophysical events can be identified more clearly. The effectiveness of the proposed method is tested using synthetic benchmark signals, laboratory data and the field dataset.

  6. Characterization of a novel splicing variant in the RAPTOR gene

    International Nuclear Information System (INIS)

    Sun Chang; Southard, Catherine; Di Rienzo, Anna

    2009-01-01

    The mammalian target of rapamycin (mTOR) plays an essential role in the regulation of cell growth, proliferation and apoptosis. Raptor, the regulatory associated protein of mTOR, is an important member in this signaling pathway. In the present report, we identified and characterized a novel splicing variant of this gene, RAPTOR v 2, in which exons 14-17, 474 bp in total, are omitted from the mRNA. This deletion does not change the open reading frame, but causes a nearly complete absence of HEAT repeats, which were shown to be involved in the binding of mTOR substrates. Real time PCR performed on 48 different human tissues demonstrated the ubiquitous presence of this splice variant. Quantification of mRNA levels in lymphoblastoid cell lines (LCL) from 56 unrelated HapMap individuals revealed that the expression of this splicing form is quite variable. One synonymous SNP, rs2289759 in exon 14, was predicted by ESEfinder to cause a significant gain/loss of SRp55 and/or SF2/ASF binding sites, and thus potentially influence splicing. This prediction was confirmed by linear regression analysis between the ratio of RAPTOR v 2 to total RAPTOR mRNA levels and the SNP genotype in the above 56 individuals (r = 0.281 and P = 0.036). Moreover, the functional evaluation indicated that this splicing isoform is expected to retain the ability to bind mTOR, but is unlikely to bind mTOR substrates, hence affecting signal transduction and further cell proliferation

  7. Getting from A to B-exploring the activation motifs of the class B adhesion G protein-coupled receptor subfamily G member 4/GPR112

    DEFF Research Database (Denmark)

    Cornelia Peeters, Miriam; Mos, Iris; Lenselink, Eelke B

    2016-01-01

    The adhesion G protein-coupled receptors (ADGRs/class B2 G protein-coupled receptors) constitute an ancient family of G protein-coupled receptors that have recently been demonstrated to play important roles in cellular and developmental processes. Here, we describe a first insight...... into the structure-function relationship of ADGRs using the family member ADGR subfamily G member 4 (ADGRG4)/GPR112 as a model receptor. In a bioinformatics approach, we compared conserved, functional elements of the well-characterized class A and class B1 secretin-like G protein-coupled receptors with the ADGRs. We...... identified several potential equivalent motifs and subjected those to mutational analysis. The importance of the mutated residues was evaluated by examining their effect on the high constitutive activity of the N-terminally truncated ADGRG4/GPR112 in a 1-receptor-1-G protein Saccharomyces cerevisiae...

  8. Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

    Directory of Open Access Journals (Sweden)

    Mi Young Yang

    2013-05-01

    Full Text Available GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

  9. Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function

    International Nuclear Information System (INIS)

    Asai, Kengo; Platt, Craig; Cochrane, Alan

    2003-01-01

    The control of HIV-1 viral RNA splicing and transport plays an important role in the successful replication of the virus. Previous studies have identified both an exon splicing enhancer (ESE) and a bipartite exon splicing silencer (ESS3a and ESS3b) within the terminal exon of HIV-1 that are involved in modulating both splicing and Rev-mediated export of viral RNA. To define the mechanism of ESS3a function, experiments were carried out to better define the cis and trans components required for ESS3a activity. Mutations throughout the 30-nt element resulted in partial loss of ESS function. Combining mutations was found to have an additive effect, suggesting the presence of multiple binding sites. Analysis of interacting factors identified hnRNP A1 as one component of the complex that modulates ESS3a activity. However, subsequent binding analyses determined that hnRNP A1 interacts with only one portion of ESS3a, suggesting the involvement of another host factor. Parallel analysis of the effect of the mutations on Rev-mediated export determined that there is not a direct correlation between the effect of the mutations on splicing and RNA transport. Consistent with this hypothesis, replacement of ESS3a with consensus hnRNP A1 binding sites was found to be insufficient to block Rev-mediated RNA export

  10. HOLLYWOOD: a comparative relational database of alternative splicing.

    Science.gov (United States)

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  11. GPR Activities in Italy: a Review

    Science.gov (United States)

    Tosti, Fabio; Ambrosanio, Michele; Battaglia, Enzo; Bianchini Ciampoli, Luca; De Carlo, Lorenzo; Matera, Loredana; Prontera, Santo; Sileo, Maria

    2015-04-01

    the ASTM D 4748-98 and the ASTM D 6432-99 dealing with, respectively, thicknesses evaluation of bound layers in road pavements, and equipment, field procedures and data-interpretation for the electromagnetic evaluation of subsurface materials, it has to be noted that the Italian body of laws and rules tackles the ground-penetrating radar applications under an indirect and partial approach. Despite of such situation, national guidelines concerning utilities-detection activities as well as other theoretical and practical guidelines established by the major Italian private enterprises on this field can be also considered highly relevant. Moreover, a further focus on the activities and main devices of the major Italian ground-penetrating radar manufacturers have been thoroughly described. Under a research and innovation perspective, the most important test sites, such as the site of the University of Salento to reconstruct archaeological and urban subsurface scenarios have been listed along with the main advances reached in integrating ground-penetrating radar with other non-destructive techniques, to inform and potentially improve the possibility of new developments and collaborations. Acknowledgements - This work is a contribution from the Training School on "Civil Engineering Applications of GPR" held in Pisa, Italy, on September 22nd - 25th, 2014, within the framework of the COST Action TU1208 "Civil Engineering Applications of Ground Penetrating Radar".

  12. A medium-chain fatty acid receptor Gpr84 in zebrafish: expression pattern and roles in immune regulation.

    Science.gov (United States)

    Huang, Qiaoyan; Feng, Dong; Liu, Kai; Wang, Peng; Xiao, Hongyan; Wang, Ying; Zhang, Shicui; Liu, Zhenhui

    2014-08-01

    Gpr84 was recently identified as a receptor for medium-chain fatty acids, but its functions remain to be clarified. We reported the identification of a zebrafish Gpr84 homologue (zGpr84), which has a higher gene expression in the tissues of intestine, heart and liver. During embryogenesis, zGpr84 is maternally expressed and a significant increase is observed at segmentation period, and it is mainly restricted to the head region, pectoral fins, branchial arches, intestine and lateral line neuromast. Fasting or treatment with lipopolysaccharide (LPS) can induce significant up-regulation of zGpr84. We further demonstrated that zGpr84 is involved in the accumulation of lipid droplets in cells. Moreover, undecanoic acid (UA) can amplify LPS induced production of the proinflammatory cytokine IL-12 p40 through zGpr84, supporting the proposal that Gpr84 may play a role in directly linking fatty acid metabolism to immunological regulation. The resulting data in fish lay a foundation for a comprehensive exploration of the functions and evolution of Gpr84. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Entropic contributions to the splicing process

    International Nuclear Information System (INIS)

    Osella, Matteo; Caselle, Michele

    2009-01-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model

  14. GPR Signal Denoising and Target Extraction With the CEEMD Method

    KAUST Repository

    Li, Jing

    2015-04-17

    In this letter, we apply a time and frequency analysis method based on the complete ensemble empirical mode decomposition (CEEMD) method in ground-penetrating radar (GPR) signal processing. It decomposes the GPR signal into a sum of oscillatory components, with guaranteed positive and smoothly varying instantaneous frequencies. The key idea of this method relies on averaging the modes obtained by empirical mode decomposition (EMD) applied to several realizations of Gaussian white noise added to the original signal. It can solve the mode-mixing problem in the EMD method and improve the resolution of ensemble EMD (EEMD) when the signal has a low signal-to-noise ratio. First, we analyze the difference between the basic theory of EMD, EEMD, and CEEMD. Then, we compare the time and frequency analysis with Hilbert-Huang transform to test the results of different methods. The synthetic and real GPR data demonstrate that CEEMD promises higher spectral-spatial resolution than the other two EMD methods in GPR signal denoising and target extraction. Its decomposition is complete, with a numerically negligible error.

  15. Genetics of alternative splicing evolution during sunflower domestication.

    Science.gov (United States)

    Smith, Chris C R; Tittes, Silas; Mendieta, J Paul; Collier-Zans, Erin; Rowe, Heather C; Rieseberg, Loren H; Kane, Nolan C

    2018-06-11

    Alternative splicing enables organisms to produce the diversity of proteins necessary for multicellular life by using relatively few protein-coding genes. Although differences in splicing have been identified among divergent taxa, the shorter-term evolution of splicing is understudied. The origins of novel splice forms, and the contributions of alternative splicing to major evolutionary transitions, are largely unknown. This study used transcriptomes of wild and domesticated sunflowers to examine splice differentiation and regulation during domestication. We identified substantial splicing divergence between wild and domesticated sunflowers, mainly in the form of intron retention. Transcripts with divergent splicing were enriched for seed-development functions, suggesting that artificial selection impacted splicing patterns. Mapping of quantitative trait loci (QTLs) associated with 144 differential splicing cases revealed primarily trans -acting variation affecting splicing patterns. A large proportion of identified QTLs contain known spliceosome proteins and are associated with splicing variation in multiple genes. Examining a broader set of wild and domesticated sunflower genotypes revealed that most differential splicing patterns in domesticated sunflowers likely arose from standing variation in wild Helianthus annuus and gained frequency during the domestication process. However, several domesticate-associated splicing patterns appear to be introgressed from other Helianthus species. These results suggest that sunflower domestication involved selection on pleiotropic regulatory alleles. More generally, our findings indicate that substantial differences in isoform abundances arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.

  16. Design and Simulation of Horn Antenna Using CST Software for GPR System

    Science.gov (United States)

    Joret, Ariffuddin; Sulong, M. S.; Abdullah, M. F. L.; Madun, Aziman; Haimi Dahlan, Samsul

    2018-04-01

    Detection of underground object can be made using a GPR system. This system is classified as a non-destructive technique (NDT) where the ground areas need not to be excavated. The technique used by the GPR system is by measuring the reflection of electromagnetic wave signal produced and detected by antenna which is known as the transmitter and the receiver antenna. In this study, a GPR system was studied by means of simulation using a Horn antenna as a transceiver antenna. The electromagnetic wave signal in this simulation is produced by current signal of an antenna which having a shape of modulation of Gaussian pulse which is having spectrum from 8 GHz until 12 GHz. CST and MATLAB Software are used in this GPR system simulation. A model of a Horn antenna has been designed using the CST software before the GPR’s system simulation modeled by adding a model of background in front of the Horn antenna. The simulation results show that the output signal of the Horn antenna can be used in detecting embedded object which are made from material of wood and iron. In addition, the simulation result has successfully developed a 3D model image of the GPR system using output signal of the Horn antenna. The embedded iron object in the GPR system simulation can be seen clearly by using this 3D image.

  17. GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma

    NARCIS (Netherlands)

    Moreno, Marta; Pedrosa, Leire; Pare, Laia; Pineda, Estela; Bejarano, Leire; Martinez, Josefina; Balasubramaniyan, Veerakumar; Ezhilarasan, Ravesanker; Kallarackal, Naveen; Kim, Sung-Hak; Wang, Jia; Audia, Alessandra; Conroy, Siobhan; Marin, Mercedes; Ribalta, Teresa; Pujol, Teresa; Herreros, Antoni; Tortosa, Avelina; Mira, Helena; Alonso, Marta M.; Gomez-Manzano, Candelaria; Graus, Francesc; Sulman, Erik P.; Piao, Xianhua; Nakano, Ichiro; Prat, Aleix; Bhat, Krishna P.; de la Iglesia, Nuria

    2017-01-01

    A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and

  18. PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor

    International Nuclear Information System (INIS)

    Maita, Hiroshi; Kitaura, Hirotake; Keen, T. Jeffrey; Inglehearn, Chris F.; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M.M.

    2004-01-01

    PAP-1 is an in vitro phosphorylation target of the Pim-1 oncogene. Although PAP-1 binds to Pim-1, it is not a substrate for phosphorylation by Pim-1 in vivo. PAP-1 has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). However, RP9 is a rare disease and only two missense mutations have been described, so the report of a link between PAP-1 and RP9 was tentative. The precise cellular role of PAP-1 was also unknown at that time. We now report that PAP-1 localizes in nuclear speckles containing the splicing factor SC35 and interacts directly with another splicing factor, U2AF35. Furthermore, we used in vitro and in vivo splicing assays to show that PAP-1 has an activity, which alters the pattern of pre-mRNA splicing and that this activity is dependent on the phosphorylation state of PAP-1. We used the same splicing assay to examine the activities of two mutant forms of PAP-1 found in RP9 patients. The results showed that while one of the mutations, H137L, had no effect on splicing activity compared with that of wild-type PAP-1, the other, D170G, resulted in both a defect in splicing activity and a decreased proportion of phosphorylated PAP-1. The D170G mutation may therefore cause RP by altering splicing of retinal genes through a decrease in PAP-1 phosphorylation. These results demonstrate that PAP-1 has a role in pre-mRNA splicing and, given that three other splicing factors have been implicated in adRP, this finding provides compelling further evidence that PAP-1 is indeed the RP9 gene

  19. Overview and comparative study of GPR international standards and guidelines - COST Action TU1208

    Science.gov (United States)

    Pajewski, Lara; Marciniak, Marian; Benedetto, Andrea; Tosti, Fabio

    2016-04-01

    for Determining the Thickness of Bound Pavement Layers Using Short-Pulse Radar, ASTM International, West Conshohocken, PA, 2010, www.astm.org, DOI: 10.1520/D4748-10. Further ASTM standards exist, not focused on GPR but including useful information (details are not provided here, for brevity reasons). There are no standards in Europe, instead, guiding the GPR use for subsurface prospecting and regulating the numerous applications of this non-destructive technique. The following Radio and Telecommunications Terminal Equipment (R&TTE) directive applies to GPR equipment and allows the placing of a GPR product on the European (EU) market for sale: Directive 1999/5/EC of the European Parliament and of the Council of 9 March 1999, on radio equipment and telecommunications terminal equipment and the mutual recognition of their conformity. Official Journal of the European Union, L 91, 7.4.1999, open access on ec.europa.eu. This document will be repealed, since 13 June 2016, by the following R&TTE directive: Directive 2014/53/EU of the European Parliament and of the Council of 16 April 2014, on the harmonisation of the laws of the Member States relating to the making available on the market of radio equipment, repealing Directive 1999/5/EC. Official Journal of the European Union, L 153, 22.5.2014, open access on ec.europa. Although conformance to the R&TTE directive allows the placing of a GPR product on the market for sale, it does not give authority for its use. In order to use the equipment, in the majority of EU member countries, a license is required. The license is controlled and issued by the radio administration in each of the member countries. The Electronic Communications Committee (ECC) of the European Conference of Postal and Telecommunications Administrations (CEPT) considers and develops policies on electronic communications activities in European context, taking account of European and international legislations and regulations. There are 48 European countries

  20. Gaussian Process Regression (GPR) Representation in Predictive Model Markup Language (PMML).

    Science.gov (United States)

    Park, J; Lechevalier, D; Ak, R; Ferguson, M; Law, K H; Lee, Y-T T; Rachuri, S

    2017-01-01

    This paper describes Gaussian process regression (GPR) models presented in predictive model markup language (PMML). PMML is an extensible-markup-language (XML) -based standard language used to represent data-mining and predictive analytic models, as well as pre- and post-processed data. The previous PMML version, PMML 4.2, did not provide capabilities for representing probabilistic (stochastic) machine-learning algorithms that are widely used for constructing predictive models taking the associated uncertainties into consideration. The newly released PMML version 4.3, which includes the GPR model, provides new features: confidence bounds and distribution for the predictive estimations. Both features are needed to establish the foundation for uncertainty quantification analysis. Among various probabilistic machine-learning algorithms, GPR has been widely used for approximating a target function because of its capability of representing complex input and output relationships without predefining a set of basis functions, and predicting a target output with uncertainty quantification. GPR is being employed to various manufacturing data-analytics applications, which necessitates representing this model in a standardized form for easy and rapid employment. In this paper, we present a GPR model and its representation in PMML. Furthermore, we demonstrate a prototype using a real data set in the manufacturing domain.

  1. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David

    2007-01-01

    , and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional......, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. CONCLUSION: Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form...... of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process....

  2. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  3. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    Science.gov (United States)

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

    Science.gov (United States)

    Shinde, Mansi Y.; Sidoli, Simone; Kulej, Katarzyna; Mallory, Michael J.; Radens, Caleb M.; Reicherter, Amanda L.; Myers, Rebecca L.; Barash, Yoseph; Lynch, Kristen W.; Garcia, Benjamin A.; Klein, Peter S.

    2017-01-01

    Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. PMID:28916722

  5. The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

    Science.gov (United States)

    Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

    2015-12-04

    The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Stimulation of GPR30 increases release of EMMPRIN-containing microvesicles in human uterine epithelial cells.

    Science.gov (United States)

    Burnett, Lindsey A; Light, Mallory M; Mehrotra, Pavni; Nowak, Romana A

    2012-12-01

    Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

  7. The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse.

    Directory of Open Access Journals (Sweden)

    Helen Waller-Evans

    2010-11-01

    Full Text Available Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.

  8. The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.

    Science.gov (United States)

    Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey; Adams, Katrina L; Novitch, Bennett G; Black, Douglas L

    2015-12-24

    The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.

  9. Genome-wide survey of allele-specific splicing in humans

    Directory of Open Access Journals (Sweden)

    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array

  10. Screening for GPR101 defects in pediatric pituitary corticotropinomas.

    OpenAIRE

    Trivellin, Giampaolo(*); Correa, Ricardo R.(*); Batsis, Maria; Faucz, Fabio R.; Chittiboina, Prashant; Bjelobaba, Ivana; Larco, Darwin O.; Quezado, Martha; Daly, Adrian; Stojilkovic, Stanko S.; Wu, T. John; Beckers, Albert; Lodish, Maya; Stratakis, Constantine A.

    2016-01-01

    Cushing disease (CD) in children is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Germline or somatic mutations in genes such as MEN1, CDKIs, AIP, and USP8 have been identified in pediatric CD, but the genetic defects in a significant percentage of cases are still unknown. We investigated the orphan G protein-coupled receptor GPR101, a gene known to be involved in somatotropinomas, for its possible involvement in corticotropinomas. We performed GPR101 sequencing, ...

  11. Characterization of GPR101 transcript structure and expression patterns

    OpenAIRE

    Trivellin, Giampaolo; Bjelobaba, Ivana; Daly, Adrian F.; Larco, Darwin O.; Palmeira, Leonor; Faucz, Fabio R.; Thiry, Albert; Leal, Letícia F.; Rostomyan, Liliya; Quezado, Martha; Schernthaner-Reiter, Marie Helene; Janjic, Marija M.; Villa, Chiara; Wu, T. John; Stojilkovic, Stanko S.

    2016-01-01

    We recently showed that Xq26.3 microduplications cause X-linked acrogigantism (X-LAG). X-LAG patients mainly present with growth hormone and prolactin-secreting adenomas and share a minimal duplicated region containing at least four genes. GPR101 was the only gene highly expressed in their pituitary lesions, but little is known about its expression patterns. GPR101 transcripts were characterized in human tissues by 5’-RACE and RNAseq, while the putative promoter was bioinformatically predicte...

  12. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins.

    Directory of Open Access Journals (Sweden)

    Ying Lin

    Full Text Available Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa N-intein fragment and S11 split inteins having a very small (6 aa C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100% of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ~1.7 × 10(-4 s(-1 to ~3.8 × 10(-4 s(-1, which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.

  13. Alternative RNA splicing and gastric cancer.

    Science.gov (United States)

    Li, Ying; Yuan, Yuan

    2017-07-01

    Alternative splicing (AS) linked to diseases, especially to tumors. Recently, more and more studies focused on the relationship between AS and gastric cancer (GC). This review surveyed the hot topic from four aspects: First, the common types of AS in cancer, including exon skipping, intron retention, mutually exclusive exon, alternative 5 ' or 3' splice site, alternative first or last exon and alternative 3' untranslated regions. Second, basic mechanisms of AS and its relationship with cancer. RNA splicing in eukaryotes follows the GT-AG rule by both cis-elements and trans-acting factors regulatory. Through RNA splicing, different proteins with different forms and functions can be produced and may be associated with carcinogenesis. Third, AS types of GC-related genes and their splicing variants. In this paper, we listed 10 common genes with AS and illustrated its possible molecular mechanisms owing to genetic variation (mutation and /or polymorphism). Fourth, the splicing variants of GC-associated genes and gastric carcinogenesis, invasion and metastasis. Many studies have found that the different splicing variants of the same gene are differentially expressed in GC and its precancerous diseases, suggesting AS has important implications in GC development. Taking together, this review highlighted the role of AS and splicing variants in the process of GC. We hope that this is not only beneficial to advances in the study field of GC, but also can provide valuable information to other similar tumor research.Although we already know some gene splicing and splicing variants play an important role in the development of GC, but many phenomena and mechanisms are still unknown. For example, how the tumor microenvironment and signal transduction pathway effect the forming and function of AS? Unfortunately, this review did not cover the contents because the current study is limited. It is no doubt that clarifying the phenomena and mechanisms of these unknown may help to reveal

  14. Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91

    DEFF Research Database (Denmark)

    Trauelsen, Mette; Rexen Ulven, Elisabeth; Hjorth, Siv A

    2017-01-01

    OBJECTIVE: Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. METHODS AND RESULTS: Here we first determined that the cis conformation of the succinate...

  15. Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84

    OpenAIRE

    Mahmud, Zobaer Al; Jenkins, Laura; Ulven, Trond; Labéguère, Frédéric; Gosmini, Romain; De Vos, Steve; Hudson, Brian D.; Tikhonova, Irina G.; Milligan, Graeme

    2017-01-01

    Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predic...

  16. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  17. Precast concrete unit assessment through GPR survey and FDTD modelling

    Science.gov (United States)

    Campo, Davide

    2017-04-01

    Precast concrete elements are widely used within United Kingdom house building offering ease in assembly and added values as structural integrity, sound and thermal insulation; most common concrete components include walls, beams, floors, panels, lintels, stairs, etc. The lack of respect of the manufacturer instruction during assembling, however, may induce cracking and short/long term loss of bearing capacity. GPR is a well-established not destructive technique employed in the assessment of structural elements because of real-time imaging, quickness of data collecting and ability to discriminate finest structural details. In this work, GPR has been used to investigate two different precast elements: precast reinforced concrete planks constituting the roof slab of a school and precast wood-cement blocks with insulation material pre-fitted used to build a perimeter wall of a private building. Visible cracks affected both constructions. For the assessment surveys, a GSSI 2.0 GHz GPR antenna has been used because of the high resolution required and the small size of the antenna case (155 by 90 by 105mm) enabling scanning up to 45mm from any obstruction. Finite Difference Time Domain (FDTD) numerical modelling was also performed to build a scenario of the expected GPR signal response for a preliminary real-time interpretation and to help solve uncertainties due to complex reflection patterns: simulated radargrams were built using Reflex Software v. 8.2, reproducing the same GPR pulse used for the surveys in terms of wavelet, nominal frequency, sample frequency and time window. Model geometries were derived from the design projects available both for the planks and the blocks; the electromagnetic properties of the materials (concrete, reinforcing bars, air-filled void, insulation and wooden concrete) were inferred from both values reported in literature and a preliminary interpretation of radargrams where internal layer interfaces were clearly recognizable and

  18. Alternative Splicing as a Target for Cancer Treatment.

    Science.gov (United States)

    Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

    2018-02-11

    Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

  19. Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

    Science.gov (United States)

    Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

    2016-01-01

    The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

  20. A five' splice-region G → C mutation in exon 1 of the human β-globin gene inhibits pre-mRNA splicing: A mechanism for β+-thalassemia

    International Nuclear Information System (INIS)

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M.; Gattoni, R.; Stevenin, J.; Chibani, J.

    1989-01-01

    The authors have characterized a Mediterranean β-thalassemia allele containing a sequence change at codon 30 that alters both β-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G → C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg → Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5

  1. Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

    Science.gov (United States)

    Fu, Xiang-Dong

    2017-04-01

    EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

  2. A study of alternative splicing in the pig

    Directory of Open Access Journals (Sweden)

    Jørgensen Claus B

    2010-05-01

    Full Text Available Abstract Background Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence. Results The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR. Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific. Conclusions In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue

  3. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

  4. Characterizing fractures and shear zones in crystalline rock using seismic and GPR methods

    Science.gov (United States)

    Doetsch, Joseph; Jordi, Claudio; Laaksonlaita, Niko; Gischig, Valentin; Schmelzbach, Cedric; Maurer, Hansruedi

    2016-04-01

    Understanding the natural or artificially created hydraulic conductivity of a rock mass is critical for the successful exploitation of enhanced geothermal systems (EGS). The hydraulic response of fractured crystalline rock is largely governed by the spatial organization of permeable fractures. Defining the 3D geometry of these fractures and their connectivity is extremely challenging, because fractures can only be observed directly at their intersections with tunnels or boreholes. Borehole-based and tunnel-based ground-penetrating radar (GPR) and seismic measurements have the potential to image fractures and other heterogeneities between and around boreholes and tunnels, and to monitor subtle time-lapse changes in great detail. We present the analysis of data acquired in the Grimsel rock laboratory as part of the In-situ Stimulation and Circulation (ISC) experiment, in which a series of stimulation experiments have been and will be performed. The experiments in the granitic rock range from hydraulic fracturing to controlled fault-slip experiments. The aim is to obtain a better understanding of coupled seismo-hydro-mechanical processes associated with high-pressure fluid injections in crystalline rocks and their impact on permeability creation and enhancement. GPR and seismic data have been recorded to improve the geological model and characterize permeable fractures and shear zones. The acquired and processed data include reflection GPR profiles measured from tunnel walls, single-borehole GPR images, and borehole-to-borehole and tunnel-to-tunnel seismic and GPR tomograms. The reflection GPR data reveal the geometry of shear zones up to a distance of 30 m from the tunnels and boreholes, but the interpretation is complicated by the geometrical ambiguity around tunnels and boreholes and by spurious reflections from man-made structures such as boreholes. The GPR and seismic traveltime tomography results reveal brittle fractured rock between two ductile shear zones. The

  5. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  6. Two new splice variants in porcine PPARGC1A

    Directory of Open Access Journals (Sweden)

    Peelman Luc J

    2008-12-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers. Findings This led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa, of which the first 291 aa would be the same compared to the complete protein (796 aa. Conclusion Considering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.

  7. Some examples of GPR prospecting for monitoring of the monumental heritage

    International Nuclear Information System (INIS)

    Masini, Nicola; Persico, Raffaele; Rizzo, Enzo

    2010-01-01

    In this paper three case histories of ground penetrating radar (GPR) for the monitoring of historical buildings are presented. They aim to present the specific valence of the GPR in the field of the diagnostic of historical buildings, which is a promising field of research, due to the increasing awareness of the relevance (even economic) of the cultural heritage. The presented GPR prospecting cases have been performed on three different constructive elements typical of historical buildings (a wall, a masonry pillar and a marble column) in order to be the answer to different problems such as the characterization of the masonry, the detection of cracks and the imaging of metallic reinforcement bars

  8. Identification of a novel function of CX-4945 as a splicing regulator.

    Directory of Open Access Journals (Sweden)

    Hyeongki Kim

    Full Text Available Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2 and a molecule currently in clinical trials (Phase II for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3-90 nM was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.

  9. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Directory of Open Access Journals (Sweden)

    Pavan Kumar P

    2014-03-01

    Full Text Available TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  10. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Science.gov (United States)

    Kumar P, Pavan; Franklin, Sarah; Emechebe, Uchenna; Hu, Hao; Moore, Barry; Lehman, Chris; Yandell, Mark; Moon, Anne M

    2014-03-01

    TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  11. Deep Ground Penetrating Radar (GPR) WIPL-D Models of Buried Sub-Surface Radiators

    National Research Council Canada - National Science Library

    Norgard, John D; Wicks, Michael C; Musselman, Randy L

    2005-01-01

    .... A new Ground Penetrating Radar (GPR) concept is proposed in this paper to use subsurface radiators, delivered as earth penetrating non-explosive, electronic e-bombs, as the source of strong radiated transmissions for GPR experiments...

  12. Analysis of Maxi-K alpha subunit splice variants in human myometrium

    Directory of Open Access Journals (Sweden)

    Morrison John J

    2004-09-01

    Full Text Available Abstract Background Large-conductance, calcium-activated potassium (Maxi-K channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. Methods Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP, and at Caesarean section, at elective (pregnant not-in-labour, PNL and intrapartum (pregnant in-labour, PL procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. Results RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. Conclusions Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at

  13. Language study on Spliced Semigraph using Folding techniques

    Science.gov (United States)

    Thiagarajan, K.; Padmashree, J.

    2018-04-01

    In this paper, we proposed algorithm to identify cut vertices and cut edges for n-Cut Spliced Semigraph and splicing the n-Cut Spliced Semigraph using cut vertices else cut edges or combination of cut vertex and cut edge and applying sequence of folding to the spliced semigraph to obtain the semigraph quadruple η(S)=(2, 1, 1, 1). We observed that the splicing and folding using both cut vertices and cut edges is applicable only for n-Cut Spliced Semigraph where n > 2. Also, we transformed the spliced semigraph into tree structure and studied the language for the semigraph with n+2 vertices and n+1 semivertices using Depth First Edge Sequence algorithm and obtain the language structure with sequence of alphabet ‘a’ and ‘b’.

  14. Two splice variants of the bovine lactoferrin gene identified in Staphylococcus aureus isolated from mastitis in dairy cattle.

    Science.gov (United States)

    Huang, J M; Wang, Z Y; Ju, Z H; Wang, C F; Li, Q L; Sun, T; Hou, Q L; Hang, S Q; Hou, M H; Zhong, J F

    2011-12-21

    Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

  15. A novel loss-of-function mutation in GPR54/KISS1R leads to hypogonadotropic hypogonadism in a highly consanguineous family.

    Science.gov (United States)

    Nimri, Revital; Lebenthal, Yael; Lazar, Liora; Chevrier, Lucie; Phillip, Moshe; Bar, Meytal; Hernandez-Mora, Eva; de Roux, Nicolas; Gat-Yablonski, Galia

    2011-03-01

    The G protein-coupled receptor 54 (GPR54), the kisspeptin receptor, is essential for stimulation of GnRH secretion and induction of puberty. Recently loss-of-function mutations of the GPR54 have been implicated as a cause of isolated idiopathic hypogonadotropic hypogonadism (IHH). The objective of the study was to identify the genetic cause of IHH in a consanguineous pedigree and to characterize the phenotypic features from infancy through early adulthood. In six patients with normosmic IHH belonging to two families of Israeli Muslim-Arab origin highly related to one another, DNA was analyzed for mutations in the GnRHR and GPR54 genes, with functional analysis of the mutation found. The five males underwent comprehensive endocrine evaluation and were under longitudinal follow-up; the one female presented in early adulthood. A new homozygous mutation (c.T815C) in GPR54 leading to a phenylalanine substitution by serine (p.F272S) was detected in all patients. Functional analysis showed an almost complete inhibition of kisspeptin-induced GPR54 signaling and a dramatic decrease of the mutated receptor expression at the cell surface. The males exhibited the same clinical features from infancy to adulthood, characterized by cryptorchidism, a relatively short penis, and no spontaneous pubertal development. The female patient presented at 18 yr with impuberism and primary amenorrhea. Repeated stimulation tests demonstrated complete gonadotropin deficiency throughout follow-up. A novel loss-of-function mutation (p.F272S) in the GPR54 gene is associated with familial normosmic IHH. Underdeveloped external genitalia and impuberism point to the major role of GPR54 in the activation of the gonadotropic axis from intrauterine life to adulthood.

  16. GPR Image and Signal Processing for Pavement and Road Monitoring on Android Smartphones and Tablets

    Science.gov (United States)

    Benedetto, Francesco; Benedetto, Andrea; Tedeschi, Antonio

    2014-05-01

    Ground Penetrating Radar (GPR) is a geophysical method that uses radar pulses to image the subsurface. This non-destructive method uses electromagnetic radiation and detects the reflected signals from subsurface structures. It can detect objects, changes in material, and voids and cracks. GPR has many applications in a number of fields. In the field of civil engineering one of the most advanced technologies used for road pavement monitoring is based on the deployment of advanced GPR systems. One of the most relevant causes of road pavement damage is often referable to water intrusion in structural layers. In this context, GPR has been recently proposed as a method to estimate moisture content in a porous medium without preventive calibration. Hence, the development of methods to obtain an estimate of the moisture content is a crucial research field involving economic, social and strategic aspects in road safety for a great number of public and private Agencies. In particular, a recent new approach was proposed to estimate moisture content in a porous medium basing on the theory of Rayleigh scattering, showing a shift of the frequency peak of the GPR spectrum towards lower frequencies as the moisture content increases in the soil. Addressing some of these issues, this work proposes a mobile application, for smartphones and tablets, for GPR image and signal processing. Our application has been designed for the Android mobile operating system, since it is open source and android mobile platforms are selling the most smartphones in the world (2013). The GPR map can be displayed in black/white or color and the user can zoom and navigate into the image. The map can be loaded in two different ways: from the local memory of the portable device or from a remote server. This latter possibility can be very useful for real-time and mobile monitoring of road and pavement inspection. In addition, the application allows analyzing the GPR data also in the frequency domain. It is

  17. The Application of GPR in Florida for Detecting Forensic Burials

    Energy Technology Data Exchange (ETDEWEB)

    S. K. Koppenjan; J. J. Schultz; S. Ono; H. Lee

    2003-01-01

    A study was performed at the University of Florida to measure ground penetrating radar(GPR) performance for detecting forensic burials. In controlled scenarios, 24 burials were constructed with pig cadavers. Two soils were utilized to represent two of the most common soil orders in Florida: an Entisol and an Ultisol. Graves were monitored on a monthly basis for time periods up to 21 months with grid data acquired with pulsed and swept-frequency GPR systems incorporating several different frequency antennas. A small subset of the graves was excavated to assess decomposition and relate to the GPR images during the test. The grave anomalies in the GPR depth profiles became less distinctive over time due to body decomposition and settling of the disturbed soil (backfill) as it compacted. Soil type was a major factor. Grave anomalies became more difficult to recognize over time for deep targets that were within clay. Forensic targets that were in sandy soil were recognized for the duration of this study. Time elapsed imagery will be presented to elucidate the changes, or lack thereof, of grave anomalies over the duration of this study. Further analysis was performed using Synthetic Aperture Radar (SAR) reconstruction of images in 2-D and 3-D.

  18. GPR capabilities for ice thickness sampling of low salinity ice and for detecting oil in ice

    Energy Technology Data Exchange (ETDEWEB)

    Lalumiere, Louis [Sensors by Design Ltd. (Canada)

    2011-07-01

    This report discusses the performance and capabilities test of two airborne ground-penetrating radar (GPR) systems of the Bedford Institute of Oceanography (BIO), Noggin 1000 and Noggin 500, for monitoring low salinity snow and ice properties which was used to measure the thickness of brackish ice on Lake Melville in Labrador and on a tidal river in Prince Edward Island. The work of other researchers is documented and the measurement techniques proposed are compared to the actual GPR approach. Different plots of GPR data taken over snow and freshwater ice and over ice with changing salinity are discussed. An interpretation of brackish ice GPR plots done by the Noggin 1000 and Noggin 500 systems is given based on resolution criterion. Additionally, the capability of the BIO helicopter-borne GPR to detect oil-in-ice has been also investigated, and an opinion on the likelihood of the success of GPR as an oil-in-ice detector is given.

  19. A Forward-Looking High-Resolution GPR System

    National Research Council Canada - National Science Library

    Kositsky, Joel; Milanfar, Peyman

    1999-01-01

    A high-resolution ground penetrating radar (GPR) system was designed to help define the optimal radar parameters needed for the efficient standoff detection of buried and surface-laid antitank mines...

  20. Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome

    Directory of Open Access Journals (Sweden)

    Xuexia Zhou

    2014-12-01

    Full Text Available Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

  1. Inverse scattering and GPR data processing: an Introduction

    Science.gov (United States)

    Persico, Raffaele

    2014-05-01

    Inverse scattering and GPR data processing: an Introduction Raffaele Persico This abstract is meant to propose a brief overview of the book "Introduction to Ground Penetrating Radar: Inverse scattering and data processing", edited by Wiley Press (ISBN 9781118305003). The reason why I propose this contribution is the fact that, in spite of the large relevant literature, to the best of my knowledge it is not very common to find a text entirely devoted to the physical-mathematical aspects (a part of them, of course) of GPR data processing. Also due to this, probably a sort of gap between the GPR practice and the underlying theory has been created, and indeed we can meet practitioners convinced that the quality of the achieved results is indefinitely improvable by making narrower the spatial step of the data, or that it is desirable to have extremely directive antennas because this would improve the resolution. In order to provide a work hopefully able to address these and other aspects and hopefully able to give a contribution to the correction of these imprecise beliefs, a dealing from the beginning has been proposed, i.e. a sequential, relatively plane, and as much as possible self consistent, dealing starting from the Maxwell's equations and reaching the most commonly exploited migration formulas and linear inversion algorithms, both within a 2D and a 3D framework. This follows the didactic aim to provide to the reader an insight about what can be reasonably achieved and what should be reasonably done in the field and during the processing phase in order to achieve satisfying results. In particular, the reader will be hopefully made aware not only of the mathematical passages, but also of the involved approximations, the needed assumptions and the physical limits of the final algorithms. The results have been also back-upped with numerical exercises and with some experimental tests, all of which conceived on purpose for this text, and some questions with the

  2. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  3. Towards Novel Techniques for Root Phenotyping Using GPR

    Science.gov (United States)

    Kobylinski, C.; Neely, H.; Everett, M. E.; Hays, D. B.; Lewis, K.

    2017-12-01

    The ability to phenotype roots in situ would provide information for carbon sequestration potential through increased root mass, possible water-seeking strategies by plants, and generate data for plant breeders. One technique for root phenotyping is to measure differences in soil moisture and use this data to infer root presence or absence. Current technologies for soil moisture detection include electromagnetic induction and neutron moisture meters; however, ground penetrating radar (GPR) has been suggested to monitor root phenotypes. The objective of this study is to use GPR as a novel technique for detecting roots and classifying root phenotypes based on the detection of differences in dielectric permittivity in response to changes in soil water content. The study will be conducted at two sites in Texas: Thrall, TX (Burleson clay) and Lubbock, TX (Olton clay loam). Three root types will be investigated: fibrous (grain sorghum), tap root (cowpea), and mixed (9-species). Data will be collected along a 10 m linear transect in each plot with a PulseEkko GPR bi-static unit operating at a radio frequency of 500 MHz. Additionally, an EM38-MK2 survey will be performed along each transect. Soil surface moisture readings will be collected with a ML3 ThetaProbe soil moisture sensor and a neutron moisture meter will be used to obtain soil moisture measurements down to 1.2 m. Measurements will be collected every two weeks throughout the growing season. Soil properties including particle size distribution, cation exchange capacity, and bulk density will also be measured. GPR's ability to distinguish root types across soils will be assessed.

  4. Hereditary cancer genes are highly susceptible to splicing mutations

    Science.gov (United States)

    Soemedi, Rachel; Maguire, Samantha; Murray, Michael F.; Monaghan, Sean F.

    2018-01-01

    Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing. PMID:29505604

  5. Hereditary cancer genes are highly susceptible to splicing mutations.

    Directory of Open Access Journals (Sweden)

    Christy L Rhine

    2018-03-01

    Full Text Available Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77% of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36% of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing.

  6. Characterization for capillary barriers effects in a sand box test using time-lapsed GPR measurements

    Science.gov (United States)

    Kuroda, S.; Ishii, N.; Morii, T.

    2017-12-01

    Capillary barriers have been known as the method to protect subsurface regions against infiltration from soil surface. It is caused by essentially heterogeneous structure in permeability or soil physical property and produce non-uniform infiltration process then, in order to estimate the actual situation of the capillary barrier effect, the site-characterization with imaging technique like geophysical prospecting is effective. In this study, we examine the applicability of GPR to characterization for capillary barriers. We built a sand box with 90x340x90cm in which a thin high-permeable gravel layer was embedded as a capillary barrier. We conducted an infiltration test in the sand box using porous tube array for irrigation. It is expected to lead to non-uniform flow of soil water induced by capillary barrier effects. We monitored this process by various types of GPR measurements, including time-lapsed common offset profiling (COP) with multi- frequency antenna and transmission measurements like cross-borehole radar. At first, we conducted GPR common-offset survey. It could show the depth of capillary barrier in sand box. After that we conducted the infiltration test and GPR monitoring for infiltration process. GPR profiles can detect the wetting front and estimate water content change in the soil layer above the capillary barrier. From spatial change in these results we can estimate the effect of capillary barrier and the zone where the break through occur or not. Based on these results, we will discuss the applicability of GPR for monitoring the phenomena around the capillary barrier of soil. At first, we conducted GPR common-offset survey. It could show the depth of capillary barrier in sand box. After that we conducted the infiltration test and GPR monitoring for infiltration process. GPR profiles can detect the wetting front and estimate water content change in the soil layer above the capillary barrier. From spatial change in these results we can estimate the

  7. Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Suzan M Hammond

    2014-01-01

    Full Text Available Splice switching oligonucleotides (SSOs induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™” to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

  8. Identification of the first surrogate agonists for the G protein-coupled receptor GPR132

    DEFF Research Database (Denmark)

    Shehata, Mohamed A.; Christensen, Hanna Belcik; Isberg, Vignir

    2015-01-01

    GPR132 is an orphan class A G protein-coupled receptor. It has been proposed to be activated by protons and to regulate apoptosis, atherosclerosis and inflammation, but these results are still preliminary. In the current work, we designed and screened a focused compound library using a β...

  9. Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism

    DEFF Research Database (Denmark)

    Petersen, Pia Steen; Jin, Chunyu; Madsen, Andreas Nygaard

    2011-01-01

    , conceivably due to decreased energy expenditure and adipocyte lipolytic activity.-Petersen, P. S., Jin, C., Madsen, A. N., Rasmussen, M., Kuhre, R., L. Egerod, K. L., Nielsen, L. B., Schwartz. T. W., Holst, B. Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism....

  10. A novel protein-protein interaction in the RES (REtention and Splicing) complex.

    Science.gov (United States)

    Tripsianes, Konstantinos; Friberg, Anders; Barrandon, Charlotte; Brooks, Mark; van Tilbeurgh, Herman; Seraphin, Bertrand; Sattler, Michael

    2014-10-10

    The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Single molecule analysis of c-myb alternative splicing reveals novel classifiers for precursor B-ALL.

    Directory of Open Access Journals (Sweden)

    Ye E Zhou

    Full Text Available The c-Myb transcription factor, a key regulator of proliferation and differentiation in hematopoietic and other cell types, has an N-terminal DNA binding domain and a large C-terminal domain responsible for transcriptional activation, negative regulation and determining target gene specificity. Overexpression and rearrangement of the c-myb gene (MYB has been reported in some patients with leukemias and other types of cancers, implicating activated alleles of c-myb in the development of human tumors. Alternative RNA splicing can produce variants of c-myb with qualitatively distinct transcriptional activities that may be involved in transformation and leukemogenesis. Here, by performing a detailed, single molecule assay we found that c-myb alternative RNA splicing was elevated and much more complex in leukemia samples than in cell lines or CD34+ hematopoietic progenitor cells from normal donors. The results revealed that leukemia samples express more than 60 different c-myb splice variants, most of which have multiple alternative splicing events and were not detectable by conventional microarray or PCR approaches. For example, the single molecule assay detected 21 and 22 splice variants containing the 9B and 9S exons, respectively, most of which encoded unexpected variant forms of c-Myb protein. Furthermore, the detailed analysis identified some splice variants whose expression correlated with poor survival in a small cohort of precursor B-ALL samples. Our findings indicate that single molecule assays can reveal complexities in c-myb alternative splicing that have potential as novel biomarkers and could help explain the role of c-Myb variants in the development of human leukemia.

  12. l-3,4-Dihydroxyphenylalanine induces ptosis through a GPR143-independent mechanism in mice

    Directory of Open Access Journals (Sweden)

    Suguru Ueda

    2016-09-01

    Full Text Available Through its conversion to dopamine by aromatic l-amino acid decarboxylase (AADC, l-3,4-dihydroxyphenylalanine (l-DOPA replenishes depleted brain dopamine in Parkinson's disease patients. We recently identified GPR143 as a candidate receptor for l-DOPA. In this study, we investigated the behavioral actions of l-DOPA in wild type (wt and Gpr143-deficient mice. l-DOPA dose-dependently (10–100 mg/kg, i.p. induced ptosis under treatment with 3-hydroxybenzylhydrazine, a centrally acting AADC inhibitor. This effect was not mimicked by 3-O-methyldopa. l-DOPA-induced ptosis in Gpr143-deficient mice to a similar extent as in wt mice. These results suggest that l-DOPA induces ptosis in a GPR143-independent fashion in mice.

  13. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage.

  14. Thermopriming Triggers Splicing Memory in Arabidopsis

    KAUST Repository

    Ling, Yu

    2018-02-20

    Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat shock memory and the role of priming in Arabidopsisthaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat stress responses in plants and other organisms as many of the key components of heat shock responses are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

  15. A Functional Assay for GPR55: Envision Protocol.

    Science.gov (United States)

    Anavi-Goffer, Sharon; Ross, Ruth A

    2016-01-01

    AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.

  16. cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing

    International Nuclear Information System (INIS)

    Liao, Huey-Jane; Baker, Carl C.; Princler, Gerald L.; Derse, David

    2004-01-01

    Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping

  17. Investigation of the GPR39 zinc receptor following inhibition of monoaminergic neurotransmission and potentialization of glutamatergic neurotransmission

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Gaweł, Magdalena; Librowski, Tadeusz

    2015-01-01

    Zinc can regulate neural function in the brain via the GPR39 receptor. In the present study we investigated whether inhibition of serotonin, noradrenaline and dopamine synthesis and potentialization of glutamate, via administration of p-chlorophenylalanine (pCPA), α-methyl-p-tyrosine (αMT) and N......-methyl-d-aspartatic acid (NMDA), respectively, would cause changes in GPR39 levels. Western blot analysis showed GPR39 up-regulation following 3-day administration of αMT and NMDA in the frontal cortex, and GPR39 down-regulation following 10-day administration of pCPA, αMT, and NMDA in the hippocampus of CD-1 mice....... There were no changes in serum zinc levels. Additionally, we investigated tryptophan, tyrosine and glutamate concentrations in the hippocampus and frontal cortex of GPR39 knockout (GPR39 KO) mice. Liquid chromatography-mass spectrometry (LC-MS) showed a significant decrease in tryptophan and tyrosine...

  18. The hnRNP 2H9 gene, which is involved in the splicing reaction, is a multiply spliced gene

    DEFF Research Database (Denmark)

    Honoré, B

    2000-01-01

    The hnRNP 2H9 gene products are involved in the splicing process and participate in early heat shock-induced splicing arrest. By combining low/high stringency hybridisation, database search, Northern and Western blotting it is shown that the gene is alternatively spliced into at least six...

  19. Traceless splicing enabled by substrate-induced activation of the Nostoc punctiforme Npu DnaE intein after mutation of a catalytic cysteine to serine.

    Science.gov (United States)

    Cheriyan, Manoj; Chan, Siu-Hong; Perler, Francine

    2014-12-12

    Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices. Copyright © 2014. Published by Elsevier Ltd.

  20. Advanced GPR imaging of sedimentary features: integrated attribute analysis applied to sand dunes

    Science.gov (United States)

    Zhao, Wenke; Forte, Emanuele; Fontolan, Giorgio; Pipan, Michele

    2018-04-01

    We evaluate the applicability and the effectiveness of integrated GPR attribute analysis to image the internal sedimentary features of the Piscinas Dunes, SW Sardinia, Italy. The main objective is to explore the limits of GPR techniques to study sediment-bodies geometry and to provide a non-invasive high-resolution characterization of the different subsurface domains of dune architecture. On such purpose, we exploit the high-quality Piscinas data-set to extract and test different attributes of the GPR trace. Composite displays of multi-attributes related to amplitude, frequency, similarity and textural features are displayed with overlays and RGB mixed models. A multi-attribute comparative analysis is used to characterize different radar facies to better understand the characteristics of internal reflection patterns. The results demonstrate that the proposed integrated GPR attribute analysis can provide enhanced information about the spatial distribution of sediment bodies, allowing an enhanced and more constrained data interpretation.

  1. Combined application of GPR and ERT for the assessment of a wall structure at the Heptapyrgion fortress (Thessaloniki, Greece)

    Science.gov (United States)

    Angelis, Dimitrios; Tsourlos, Panagiotis; Tsokas, Gregory; Vargemezis, George; Zacharopoulou, Georgia; Power, Christopher

    2018-05-01

    Non-destructive investigation of monuments can be an extremely valuable tool to evaluate potential structural defects and assist in developing any restoration plans. In this work, both Ground Penetrating Radar (GPR) and Electrical Resistivity Tomography (ERT) techniques were applied to a tower wall of the Heptapyrgion fortress located in Thessaloniki, Greece, which was facing significant moisture problems. GPR cross sections, mainly obtained with a 500 MHz centre frequency antenna, and ERT profiles were collected along the same survey grid on the tower wall. The gprMax numerical solver was used for the GPR forward modelling. In addition, an auxiliary program was used to design and import into gprMax complicated structures and this allowed to simulate more realistically the wall defects and moisture. The GPR simulator was used to assess and optimize the field data acquisition and processing parameters, and to assist in interpreting the GPR cross sections. The ERT sections were inverted as individual 2D lines and also, as a full 3D dataset. The final GPR and ERT data were jointly interpreted in view of the studied problem as results of both methods are highly correlated. A high moisture content area at the eastern part of the wall was identified in both GPR and ERT data, along with the interface between different phases of construction. Through the GPR data we were also able to delineate possible structural defects (cracks, small voids) which was not possible with just using the ERT data. Furthermore, a very good matching was evident between the simulated GPR modelling results incorporating field-interpreted features, and the actual field GPR results, thereby validating the proposed data interpretation. The overall survey and modelling approach produces results that are in a very good agreement between them and proved very useful in accessing the wall structure.

  2. Novel Agonist Bioisosteres and Common Structure-Activity Relationships for The Orphan G Protein-Coupled Receptor GPR139

    DEFF Research Database (Denmark)

    Shehata, Mohamed A; Jensen, Anne Cathrine Nøhr; Lissa, Delphine

    2016-01-01

    GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression levels in the hypothalamus and striatum, regions regulating metabolism and locomotion, respectively, and has therefore been suggested as a potential target for obesity...

  3. The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

    Science.gov (United States)

    Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

    2010-11-01

    G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

  4. Alternative Splicing Control of Abiotic Stress Responses.

    Science.gov (United States)

    Laloum, Tom; Martín, Guiomar; Duque, Paula

    2018-02-01

    Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. GPR158, an orphan member of G protein-coupled receptor Family C: glucocorticoid-stimulated expression and novel nuclear role.

    Science.gov (United States)

    Patel, Nitin; Itakura, Tatsuo; Gonzalez, Jose M; Schwartz, Stephen G; Fini, M Elizabeth

    2013-01-01

    Members of the large G protein-coupled receptor (GPCR) clan are implicated in many physiological and disease processes, making them important therapeutic drug targets. In the present study, we follow up on results of a pilot study suggesting a functional relationship between glucocorticoid (GC)-induced ocular hypertension and GPR158, one of three orphan members of the GPCR Family C. GC treatment increases levels of GPR158 mRNA and protein through transcriptional mechanisms, in cultured trabecular meshwork (TBM) cells derived from the eye's aqueous outflow pathway. Like treatment with GCs, transient overexpression of GPR158 stimulates cell proliferation, while siRNA knockdown of endogenous GPR158 has the opposite effect. Both endogenous and overexpressed GPR158 show an unusual subcellular localization pattern, being found almost entirely in the nucleus. However, when cells are treated with inhibitors of clathrin-mediated endocytosis, GPR158 is shifted to the plasma membrane. Mutation of a bipartite nuclear localization signal (NLS) in the 8(th) helix also shifts GPR158 out of the nucleus, but in this case the protein is found in vesicles localized in the cytoplasm. These results suggest that newly synthesized GPR158 first traffics to the plasma membrane, where it rapidly undergoes endocytosis and translocation to the nucleus. Significantly, mutation of the NLS abrogates GPR158-mediated enhancement of cell proliferation, indicating a functional requirement for nuclear localization. GPR158 overexpression upregulates levels of the cell cycle regulator cyclin D1, but mutation of the NLS reverses this. Overexpression of GPR158 enhances the barrier function of a TBM cell monolayer, which is associated with an increase in the levels of tight junction proteins ZO-1 and occludin, similar to reported studies on GC treatment. Regulated paracellular permeability controls aqueous outflow facility in vivo. Since GCs stimulate GPR158 expression, the result is consistent with a

  6. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

    Directory of Open Access Journals (Sweden)

    Delphine Laustriat

    2015-01-01

    Full Text Available Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1, a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

  7. Capacity of columns with splice imperfections

    International Nuclear Information System (INIS)

    Popov, E.P.; Stephen, R.M.

    1977-01-01

    To study the behavior of spliced columns subjected to tensile forces simulating situations which may develop in an earthquake, all of the spliced specimens were tested to failure in tension after first having been subjected to large compressive loads. The results of these tests indicate that the lack of perfect contact at compression splices of columns may not be important, provided that the gaps are shimmed and welding is used to maintain the sections in alignment

  8. Identification of genome-wide non-canonical spliced regions and analysis of biological functions for spliced sequences using Read-Split-Fly.

    Science.gov (United States)

    Bai, Yongsheng; Kinne, Jeff; Ding, Lizhong; Rath, Ethan C; Cox, Aaron; Naidu, Siva Dharman

    2017-10-03

    It is generally thought that most canonical or non-canonical splicing events involving U2- and U12 spliceosomes occur within nuclear pre-mRNAs. However, the question of whether at least some U12-type splicing occurs in the cytoplasm is still unclear. In recent years next-generation sequencing technologies have revolutionized the field. The "Read-Split-Walk" (RSW) and "Read-Split-Run" (RSR) methods were developed to identify genome-wide non-canonical spliced regions including special events occurring in cytoplasm. As the significant amount of genome/transcriptome data such as, Encyclopedia of DNA Elements (ENCODE) project, have been generated, we have advanced a newer more memory-efficient version of the algorithm, "Read-Split-Fly" (RSF), which can detect non-canonical spliced regions with higher sensitivity and improved speed. The RSF algorithm also outputs the spliced sequences for further downstream biological function analysis. We used open access ENCODE project RNA-Seq data to search spliced intron sequences against the U12-type spliced intron sequence database to examine whether some events could occur as potential signatures of U12-type splicing. The check was performed by searching spliced sequences against 5'ss and 3'ss sequences from the well-known orthologous U12-type spliceosomal intron database U12DB. Preliminary results of searching 70 ENCODE samples indicated that the presence of 5'ss with U12-type signature is more frequent than U2-type and prevalent in non-canonical junctions reported by RSF. The selected spliced sequences have also been further studied using miRBase to elucidate their functionality. Preliminary results from 70 samples of ENCODE datasets show that several miRNAs are prevalent in studied ENCODE samples. Two of these are associated with many diseases as suggested in the literature. Specifically, hsa-miR-1273 and hsa-miR-548 are associated with many diseases and cancers. Our RSF pipeline is able to detect many possible junctions

  9. GPR identification of voids inside concrete based on the support vector machine algorithm

    International Nuclear Information System (INIS)

    Xie, Xiongyao; Li, Pan; Qin, Hui; Liu, Lanbo; Nobes, David C

    2013-01-01

    Voids inside reinforced concrete, which affect structural safety, are identified from ground penetrating radar (GPR) images using a completely automatic method based on the support vector machine (SVM) algorithm. The entire process can be characterized into four steps: (1) the original SVM model is built by training synthetic GPR data generated by finite difference time domain simulation and after data preprocessing, segmentation and feature extraction. (2) The classification accuracy of different kernel functions is compared with the cross-validation method and the penalty factor (c) of the SVM and the coefficient (σ2) of kernel functions are optimized by using the grid algorithm and the genetic algorithm. (3) To test the success of classification, this model is then verified and validated by applying it to another set of synthetic GPR data. The result shows a high success rate for classification. (4) This original classifier model is finally applied to a set of real GPR data to identify and classify voids. The result is less than ideal when compared with its application to synthetic data before the original model is improved. In general, this study shows that the SVM exhibits promising performance in the GPR identification of voids inside reinforced concrete. Nevertheless, the recognition of shape and distribution of voids may need further improvement. (paper)

  10. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_patt...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  11. Evidence for a role of KISS-1/GPR54 system in decreased ...

    African Journals Online (AJOL)

    Short fasting has been shown to suppress luteinizing hormone (LH) secretion in ewes, however, the molecular mechanism underlying this phenomenon is not clear. Based on recent discovery of the crucial role of KiSS-1/GPR54 system in the central control of the GnRH axis, we hypothesized that KiSS-1/GPR54 system was ...

  12. Frequency of the GPR7 Tyr135Phe allelic variant in lean and obese subjects.

    Science.gov (United States)

    Pelosini, C; Maffei, M; Ceccarini, G; Marchi, M; Marsili, A; Galli, G; Scartabelli, G; Tamberi, A; Latrofa, F; Fierabracci, P; Vitti, P; Pinchera, A; Santini, F

    2013-10-01

    GPR7, the endogenous coupled receptor for neuropeptide B and neuropeptide W, is expressed in several regions of the central nervous system, which are involved in the regulation of feeding behavior. GPR7 affects the regulation of energy balance through a mechanism independent of leptin and melanocortin pathways. Aim of this study was to investigate whether GPR7 gene mutations can be detected in human subjects and, in that event, if they are differently distributed among lean and obese subjects. The coding region of GPR7 were sequenced in 150 obese patients and 100 normal-weight unrelated controls. Functional studies of the allelic variants were performed. One genetic GPR7 variant was found (Tyr135Phe - rs33977775) in obese subjects (13.3%) and lean control (25%). Functional studies did not reveal significant differences between the wild type and the Tyr135Phe allelic variants in their NPW-mediated capacity to inhibit forskolin-induced cAMP production. Screening of GPR7 gene mutations among lean and obese subjects revealed a Tyr135Phe allelic variant that was fairly common in the study population. As indicated by in vitro and in silico studies, this variant is unlikely to cause a functional derangement of the receptor.

  13. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    Science.gov (United States)

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-12-22

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

  14. GPR attenuation analyses using spectral ratios of primary and multiple arrivals: examples from Welsh peat bogs

    Science.gov (United States)

    Booth, A.; Carless, D.; Kulessa, B.

    2014-12-01

    Ground penetrating radar (GPR) is widely applied to qualitative and quantitative interpretation of near-surface targets. Surface deployments of GPR most widely characterise physical properties in terms of some measure of GPR wavelet velocity. Wavelet amplitude is less-often considered, potentially due to difficulties in measuring this quantity: amplitudes are distorted by the anisotropic radiation pattern of antennas, and the ringy GPR wavelet can make successive events difficult to isolate. However, amplitude loss attributes could provide a useful means of estimating the physical properties of a target. GPR energy loss is described by the bandwidth-limited quality factor Q* which, for low-loss media, is proportional to the ratio of dielectric permittivity, ɛ, and electrical conductivity, σ. Comparing the frequency content of two arrivals yields an estimate of interval Q*, but only if they are sufficiently distinct. There may be sufficient separation between a primary reflection and its long-path multiple (i.e. a 'repeat path' of the primary reflection) therefore a dataset that is rich in multiples may be suitable for robust Q* analysis. The Q* between a primary and multiple arrival describes all frequency-dependent loss mechanisms in the interval between the free-surface and the multiple-generating horizon: assuming that all reflectivity is frequency-independent, Q* can be used to estimate ɛ and/or σ. We measure Q* according to the spectral ratio method, for synthetic and real GPR datasets. Our simulations are performed using the finite-difference algorithm GprMax, and represent our example data of GPR acquisitions over peat bogs. These data are a series of 100 MHz GPR acquisitions over sites in the Brecon Beacons National Park of South Wales. The base of the bogs (the basal peat/mineral soil contact) is often a strong multiple-generating horizon. As an example, data from Waun Ddu bog show these events lagging by ~75 ns: GPR velocity is measured here at 0

  15. A liver X receptor (LXR)-β alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-β

    International Nuclear Information System (INIS)

    Hashimoto, Koshi; Ishida, Emi; Matsumoto, Shunichi; Shibusawa, Nobuyuki; Okada, Shuichi; Monden, Tsuyoshi; Satoh, Tetsurou; Yamada, Masanobu; Mori, Masatomo

    2009-01-01

    We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-β LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-β gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-β. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

  16. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    Energy Technology Data Exchange (ETDEWEB)

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  17. The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

    OpenAIRE

    Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

    2010-01-01

    G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Ex...

  18. Extracting Archaeological Feautres from GPR Surveys Conducted with Variable Soil Moisture Conditions

    Science.gov (United States)

    Morris, I. M.; Glisic, B.; Gonciar, A.

    2017-12-01

    As a common tool for subsurface archaeological prospection, ground penetrating radar (GPR) is a useful method for increasing the efficiency of archaeological excavations. Archaeological sites are often temporally and financially constrained, therefore having limited ability to reschedule surveys compromised by weather. Furthermore, electromagnetic GPR surveys are especially sensitive to variations in water content, soil type, and site-specific interference. In this work, GPR scans of a partially excavated Roman villa consisting of different construction materials and phases (limestone, andesite, brick) in central Romania are compared. Surveys were conducted with a 500 MHz GPR antenna in both dry (pre-rain event) and wet (post-rain event) conditions. Especially in time or depth slices, wet surveys present additional archaeological features that are not present or clear in the standard dry conditions, while simultaneously masking the clutter present in those scans. When dry, the limestone has a similar dielectric constant to the soil and does not provide enough contrast in electromagnetic properties for strong reflections despite the significant difference in their physical properties. Following precipitation, however, the electromagnetic properties of these two materials is dominated by their respective water content and the contrast is enhanced. For this reason, the wet surveys are particularly necessary for revealing reflections from the limestone features often invisible in dry surveys. GPR surveys conducted in variable environmental conditions provide unique archaeological information, with potential near-surface geophysical applications in nondestructive material characterization and identification.

  19. Aberrant and alternative splicing in skeletal system disease.

    Science.gov (United States)

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  20. Biased signaling of lipids and allosteric actions of synthetic molecules for GPR119

    DEFF Research Database (Denmark)

    Hassing, Helle A; Fares, Suzan; Larsen, Olav

    2016-01-01

    for 2h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100nM AR......231453. Our studies uncovering broad and biased signaling, masked constitutive activity by endogenous MAGs, and ago-allosteric properties of synthetic ligands may explain why many GPR119 drug-discovery programs have failed so far....

  1. Full-waveform inversion of GPR data for civil engineering applications

    Science.gov (United States)

    van der Kruk, Jan; Kalogeropoulos, Alexis; Hugenschmidt, Johannes; Klotzsche, Anja; Busch, Sebastian; Vereecken, Harry

    2014-05-01

    Conventional GPR ray-based techniques are often limited in their capability to image complex structures due to the pertaining approximations. Due to the increased computational power, it is becoming more easy to use modeling and inversion tools that explicitly take into account the detailed electromagnetic wave propagation characteristics. In this way, new civil engineering application avenues are opening up that enable an improved high resolution imaging of quantitative medium properties. In this contribution, we show recent developments that enable the full-waveform inversion of off-ground, on-ground and crosshole GPR data. For a successful inversion, a proper start model must be used that generates synthetic data that overlaps the measured data with at least half a wavelength. In addition, the GPR system must be calibrated such that an effective wavelet is obtained that encompasses the complexity of the GPR source and receiver antennas. Simple geometries such as horizontal layers can be described with a limited number of model parameters, which enable the use of a combined global and local search using the Simplex search algorithm. This approach has been implemented for the full-waveform inversion of off-ground and on-ground GPR data measured over horizontally layered media. In this way, an accurate 3D frequency domain forward model of Maxwell's equation can be used where the integral representation of the electric field is numerically evaluated. The full-waveform inversion (FWI) for a large number of unknowns uses gradient-based optimization methods where a 3D to 2D conversion is used to apply this method to experimental data. Off-ground GPR data, measured over homogeneous concrete specimens, were inverted using the full-waveform inversion. In contrast to traditional ray-based techniques we were able to obtain quantitative values for the permittivity and conductivity and in this way distinguish between moisture and chloride effects. For increasing chloride

  2. Applying FDEM, ERT and GPR at a site with soil contamination: A case study

    Science.gov (United States)

    Wang, Tzu-Pin; Chen, Chien-Chih; Tong, Lun-Tao; Chang, Ping-Yu; Chen, Yi-Chieh; Dong, Tien-Hsing; Liu, Hsin-Chang; Lin, Chih-Ping; Yang, Kai-Hsing; Ho, Ching-Jen; Cheng, Shih-Nan

    2015-10-01

    This study employed the combination of three methods, namely the Frequency Domain Electromagnetic (FDEM), Electrical Resistivity Tomography (ERT) and Ground Penetrating Radar (GPR) to evaluate a heavy-metal contaminated site for both pre- and post-remediation investigations. The main goals were to verify the position and the integrity of the underground storage tanks (UST), and to determine the effectiveness of remediation to ensure no contaminants remained at the site. In general, the GPR survey was effective at locating shallowly buried objects. However, due to the highly conductive nature of the heavy-metal laden sludge, the GPR signals were attenuated severely. Thus, the first attempt to use GPR in the pre-remediation investigation did not achieve the desired results and other methods were deployed. The existence of the UST and the sludge within were confirmed by ERT and the UST shape was mapped by FDEM. The principal remediation scheme was soil replacement by replacing the contaminated soil with clean silt. Based on the distinctive property differences of the contaminated soil and the clean silt, the completion of the remediation was confirmed by the differences between pre-remediation and post-remediation in GPR, ERT and FDEM results.

  3. Tracking the evolution of alternatively spliced exons within the Dscam family

    Directory of Open Access Journals (Sweden)

    Vision Todd J

    2006-02-01

    Full Text Available Abstract Background The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17, potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. Results Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee, we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248–283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. Conclusion Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays

  4. A comparison of GPR performance at various frequencies - a guide to improved survey design

    CSIR Research Space (South Africa)

    Kgarume, Thabang E

    2015-09-01

    Full Text Available This paper aims to demonstrate, through practical examples and numerical modelling, the trade-off between range and resolution in ground penetrating radar (GPR) prospecting. GPR surveys can easily fail when the target and host rock properties...

  5. Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

    OpenAIRE

    Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

    2012-01-01

    The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as wel...

  6. Tomographic site characterization using CPT, ERT, and GPR. Innovative Technology Summary Report

    International Nuclear Information System (INIS)

    None

    2000-01-01

    ARA developed a geophysical tomographic system that incorporates results from Electrical Resistivity Tomography (ERT) measurements and Ground Penetrating Radar (GPR) Tomography measurements. Both methods are useful for imaging subsurface structures and processes, however, GPR is more effective in sandy material and ERT is more effective in clayey material. CPT or drilling is used to deploy the electrodes in the subsurface

  7. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

    Directory of Open Access Journals (Sweden)

    Maik Grohmann

    Full Text Available Brain serotonin (5-HT neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2 in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1 is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

  8. Accumulation of GC donor splice signals in mammals

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2008-07-01

    Full Text Available Abstract The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing. Reviewers This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.

  9. Expression of the long-chain fatty acid receptor GPR120 in the gonadotropes of the mouse anterior pituitary gland.

    Science.gov (United States)

    Moriyama, Ryutaro; Deura, Chikaya; Imoto, Shingo; Nose, Kazuhiro; Fukushima, Nobuyuki

    2015-01-01

    G-protein-coupled receptor 120 (GPR120) has been known to be a receptor of long-chain fatty acids. Here, we investigated GPR120 expression in the mouse pituitary gland via real-time PCR, in situ hybridization, and immunohistochemistry. GPR120 mRNA was abundantly expressed in the pituitary gland of ad-lib fed animals. In situ hybridization and immunohistochemistry revealed GPR120 expression in the gonadotropes of the anterior pituitary gland, but not in thyrotropes, somatotropes, lactotropes, corticotropes, melanotropes, and the posterior pituitary gland. Furthermore, 24 h of fasting induced an increase in GPR120 mRNA expression in the pituitary gland. These results demonstrate that GPR120 in mouse pituitary gonadotropes is upregulated by fasting and that it may play a role in controlling gonadotropin secretion.

  10. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    Science.gov (United States)

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. GPR investigation to allocate the archaeological remains in Mut temple, Luxor, Upper Egypt

    Directory of Open Access Journals (Sweden)

    M.A. Atya

    2012-06-01

    Full Text Available GPR investigation has been conducted on Mut temple; to the south portion of Al-Karnak temple at the eastern bank of Luxor city. Within the survey, the GPR SIR system-10A has been used connected to 100/500 MHz antenna. The present work is oriented to allocate the buried Archaeological ruins at the site, and also to evaluate the archaeological significance of the artifacts in concern to the hydro-situation. The survey is composed of three data sets; the first set (A includes three GPR profiles located inside the temple palisade at the western bank of the holy lake, the second set (B includes four profiles distributed on the yard between Mute and Al Karnak temples, and the third set (C includes three profiles oriented to study the EW Sphinx Avenue front of Mute temple. The measured GPR data has been processed and visualized in different ways to show the infra-content of the artifacts in the buried subsurface of the temple. Furthermore, intensive mutual work and discussion with the local inspectorate at Luxor about the results would lead to detect the zones of possible findings and, as much as possible, to define their identities. A series of sectional GPR records, time slices, maps, and 3D graphs are introduced to represent the remains of Mut temple and its infrastructure.

  12. Insights into unbound-bound states of GPR142 receptor in a membrane-aqueous system using molecular dynamics simulations.

    Science.gov (United States)

    Kaushik, Aman Chandra; Sahi, Shakti

    2018-05-01

    G protein coupled receptors (GPCRs) are source machinery in signal transduction pathways and being one of the major therapeutic targets play a significant in drug discovery. GPR142, an orphan GPCR, has been implicated in the regulation of insulin, thereby having a crucial role in Type II diabetes management. Deciphering of the structures of orphan, GPCRs (O-GPCRs) offer better prospects for advancements in research in ion translocation and transduction of extracellular signals. As the crystallographic structure of GPR142 is not available in PDB, therefore, threading and ab initio-based approaches were used for 3D modeling of GPR142. Molecular dynamic simulations (900 ns) were performed on the 3D model of GPR142 and complexes of GPR142 with top five hits, obtained through virtual screening, embedded in lipid bilayer with aqueous system using OPLS force field. Compound 1, 3, and 4 may act as scaffolds for designing potential lead agonists for GPR142. The finding of GPR142 MD simulation study provides more comprehensive representation of the functional properties. The concern for Type II diabetes is increasing worldwide and successful treatment of this disease demands novel drugs with better efficacy.

  13. Global Research Patterns on Ground Penetrating Radar (GPR)

    Science.gov (United States)

    Gizzi, Fabrizio Terenzio; Leucci, Giovanni

    2018-05-01

    The article deals with the analysis of worldwide research patterns concerning ground penetrating radar (GPR) during 1995-2014. To do this, the Thomson Reuters' Science Citation Index Expanded (SCI-EXPANDED) and the Social Sciences Citation Index accessed via the Web of Science Core Collection were the two bibliographic databases taken as a reference. We pay attention to the document typology and language, the publication trend and citations, the subject categories and journals, the collaborations between authors, the productivity of the authors, the most cited articles, the countries and the institutions involved, and other hot issues. Concerning the main research subfields involving GPR use, there were five, physical-mathematical, sedimentological-stratigraphical, civil engineering/engineering geology/cultural heritage, hydrological (HD), and glaciological (GL), subfields.

  14. Alternative splicing in cancers: From aberrant regulation to new therapeutics.

    Science.gov (United States)

    Song, Xiaowei; Zeng, Zhenyu; Wei, Huanhuan; Wang, Zefeng

    2018-03-01

    Alternative splicing is one of the most common mechanisms for gene regulation in humans, and plays a vital role to increase the complexity of functional proteins. In this article, we seek to provide a general review on the relationships between alternative splicing and tumorigenesis. We briefly introduce the basic rules for regulation of alternative splicing, and discuss recent advances on dynamic regulation of alternative splicing in cancers by highlighting the roles of a variety of RNA splicing factors in tumorigenesis. We further discuss several important questions regarding the splicing of long noncoding RNAs and back-splicing of circular RNAs in cancers. Finally, we discuss the current technologies that can be used to manipulate alternative splicing and serve as potential cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Temporal monitoring of the soil freeze-thaw cycles over snow-cover land by using off-ground GPR

    KAUST Repository

    Jadoon, Khan

    2013-07-01

    We performed off-ground ground-penetrating radar (GPR) measurements over a bare agricultural field to monitor the freeze-thaw cycles over snow-cover. The GPR system consisted of a vector network analyzer combined with an off-ground monostatic horn antenna, thereby setting up an ultra-wideband stepped-frequency continuous-wave radar. Measurements were performed during nine days and the surface of the bare soil was exposed to snow fall, evaporation and precipitation as the GPR antenna was mounted 110 cm above the ground. Soil surface dielectric permittivity was retrieved using an inversion of time-domain GPR data focused on the surface reflection. The GPR forward model used combines a full-waveform solution of Maxwell\\'s equations for three-dimensional wave propagation in planar layered media together with global reflection and transmission functions to account for the antenna and its interactions with the medium. Temperature and permittivity sensors were installed at six depths to monitor the soil dynamics in the top 8 cm depth. Significant effects of soil dynamics were observed in the time-lapse GPR, temperature and permittivity data and in particular freeze and thaw events were clearly visible. A good agreement of the trend was observed between the temperature, permittivity and GPR time-lapse data with respect to five freeze-thaw cycles. The GPR-derived permittivity was in good agreement with sensor observations. The proposed method appears to be promising for the real-time mapping and monitoring of the frozen layer at the field scale. © 2013 IEEE.

  16. Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11.

    Science.gov (United States)

    Baert, Annelot; Machackova, Eva; Coene, Ilse; Cremin, Carol; Turner, Kristin; Portigal-Todd, Cheryl; Asrat, Marie Jill; Nuk, Jennifer; Mindlin, Allison; Young, Sean; MacMillan, Andree; Van Maerken, Tom; Trbusek, Martin; McKinnon, Wendy; Wood, Marie E; Foulkes, William D; Santamariña, Marta; de la Hoya, Miguel; Foretova, Lenka; Poppe, Bruce; Vral, Anne; Rosseel, Toon; De Leeneer, Kim; Vega, Ana; Claes, Kathleen B M

    2018-04-01

    For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies. © 2017 Wiley Periodicals, Inc.

  17. Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

    Directory of Open Access Journals (Sweden)

    Kathrin Davari

    2017-04-01

    Full Text Available Summary: Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP, and RNA polymerase II (RNA Pol II ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD and activation of the positive transcription elongation factor (pTEFb. Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation. : Davari et al. visualize global changes in RNA Pol II binding, transcription, splicing, and translation. T cells change their functional program by rapid de novo recruitment of RNA Pol II and coupled changes in transcription and translation. This coincides with fluctuations in RNA Pol II phosphorylation and a temporary reduction in cotranscriptional splicing. Keywords: RNA Pol II, cotranscriptional splicing, T cell activation, ribosome profiling, 4sU, H3K36, Ser-5 RNA Pol II, Ser-2 RNA Pol II, immune response, immediate-early genes

  18. The Functional Impact of Alternative Splicing in Cancer.

    Science.gov (United States)

    Climente-González, Héctor; Porta-Pardo, Eduard; Godzik, Adam; Eyras, Eduardo

    2017-08-29

    Alternative splicing changes are frequently observed in cancer and are starting to be recognized as important signatures for tumor progression and therapy. However, their functional impact and relevance to tumorigenesis remain mostly unknown. We carried out a systematic analysis to characterize the potential functional consequences of alternative splicing changes in thousands of tumor samples. This analysis revealed that a subset of alternative splicing changes affect protein domain families that are frequently mutated in tumors and potentially disrupt protein-protein interactions in cancer-related pathways. Moreover, there was a negative correlation between the number of these alternative splicing changes in a sample and the number of somatic mutations in drivers. We propose that a subset of the alternative splicing changes observed in tumors may represent independent oncogenic processes that could be relevant to explain the functional transformations in cancer, and some of them could potentially be considered alternative splicing drivers (AS drivers). Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Splicing regulatory factors, ageing and age-related disease.

    Science.gov (United States)

    Latorre, Eva; Harries, Lorna W

    2017-07-01

    Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Characterizing Watersheds with Geophysical Methods: Some uses of GPR and EMI in Hydropedological Investigations.

    Science.gov (United States)

    Doolittle, J.; Lin, H.; Jenkinson, B.; Zhou, X.

    2006-05-01

    The USDA-NRCS and its cooperators use ground-penetrating radar (GPR) and electromagnetic induction (EMI) as rapid, noninvasive tools to support soil surveys at different scales and levels of resolution. The effective use of GPR is site-specific and generally restricted to soils having low electrical conductivity (e.g., soils with low clay and soluble salt contents). In suitable soils, GPR provides high resolution data, which are used to estimate depths to soil horizons and geologic layers that restrict, redirect, and/or concentrate the flow of water through landscapes. In areas of coarse-textured soils, GPR has been used to map spatiotemporal variations in water-table depths and local ground-water flow patterns. Compared with GPR, EMI can be effectively used across a broader spectrum of soils and spatial scales, but provides lower resolution of subsurface features. EMI is used to refine and improve soil maps prepared with traditional soil survey methods. Differences in apparent conductivity (ECa) are associated with different soils and soil properties (e.g., clay, moisture and soluble salt contents). Apparent conductivity maps provide an additional layer of information, which directs soil sampling, aids the identification and delineation of some soil polygons, and enhances the quality of soil maps. More recently, these tools were used to characterize the hydropedological character of a small, steeply sloping, forested watershed. Within the watershed, EMI was used to characterize the principal soil-landscape components, and GPR was used to provide high resolution data on soil depth and layering within colluvial deposits located in swales and depressional areas.

  1. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  2. Ground penetrating radar (GPR) detects fine roots of agricultural crops in the field

    Science.gov (United States)

    Xiuwei Liu; Xuejun Dong; Qingwu Xue; Daniel I. Leskovar; John Jifon; John R. Butnor; Thomas Marek

    2018-01-01

    Aim Ground penetrating radar (GPR) as a non-invasive technique is widely used in coarse root detection. However, the applicability of the technique to detect fine roots of agricultural crops is unknown. The objective of this study was to assess the feasibility of utilizing GPR to detect fine roots in the field.

  3. DEDB: a database of Drosophila melanogaster exons in splicing graph form

    Directory of Open Access Journals (Sweden)

    Tan Tin

    2004-12-01

    Full Text Available Abstract Background A wealth of quality genomic and mRNA/EST sequences in recent years has provided the data required for large-scale genome-wide analysis of alternative splicing. We have capitalized on this by constructing a database that contains alternative splicing information organized as splicing graphs, where all transcripts arising from a single gene are collected, organized and classified. The splicing graph then serves as the basis for the classification of the various types of alternative splicing events. Description DEDB http://proline.bic.nus.edu.sg/dedb/index.html is a database of Drosophila melanogaster exons obtained from FlyBase arranged in a splicing graph form that permits the creation of simple rules allowing for the classification of alternative splicing events. Pfam domains were also mapped onto the protein sequences allowing users to access the impact of alternative splicing events on domain organization. Conclusions DEDB's catalogue of splicing graphs facilitates genome-wide classification of alternative splicing events for genome analysis. The splicing graph viewer brings together genome, transcript, protein and domain information to facilitate biologists in understanding the implications of alternative splicing.

  4. Estradiol upregulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed TMJ.

    Directory of Open Access Journals (Sweden)

    Rui-Yun Bi

    Full Text Available Temporomandibular disorders (TMDs have the highest prevalence in women of reproductive age. The role of estrogen in TMDs and especially in TMDs related pain is not fully elucidated. Voltage-gated sodium channel 1.7 (Nav1.7 plays a prominent role in pain perception and Nav1.7 in trigeminal ganglion (TG is involved in the hyperalgesia of inflamed Temporomandibular joint (TMJ. Whether estrogen could upregulate trigeminal ganglionic Nav1.7 expression to enhance hyperalgesia of inflamed TMJ remains to be explored.Estrous cycle and plasma levels of 17β-estradiol in female rats were evaluated with vaginal smear and enzyme linked immunosorbent assay, respectively. Female rats were ovariectomized and treated with 17β-estradiol at 0 μg, 20 μg and 80 μg, respectively, for 10 days. TMJ inflammation was induced using complete Freund's adjuvant. Head withdrawal thresholds and food intake were measured to evaluate the TMJ nociceptive responses. The expression of Nav1.7 in TG was examined using real-time PCR and western blot. The activity of Nav1.7 promoter was examined using luciferase reporter assay. The locations of estrogen receptors (ERα and ERβ, the G protein coupled estrogen receptor (GPR30, and Nav1.7 in TG were examined using immunohistofluorescence.Upregulation of Nav1.7 in TG and decrease in head withdrawal threshold were observed with the highest plasma 17β-estradiol in the proestrus of female rats. Ovariectomized rats treated with 80 μg 17β-estradiol showed upregulation of Nav1.7 in TG and decrease in head withdrawal threshold as compared with that of the control or ovariectomized rats treated with 0 μg or 20 μg. Moreover, 17β-estradiol dose-dependently potentiated TMJ inflammation-induced upregulation of Nav1.7 in TG and also enhanced TMJ inflammation-induced decrease of head withdrawal threshold in ovariectomized rats. In addition, the estrogen receptor antagonist, ICI 182,780, partially blocked the 17β-estradiol effect on Nav1.7

  5. Numerical modelling of GPR electromagnetic fields for locating burial sites

    Directory of Open Access Journals (Sweden)

    Carcione José M.

    2017-01-01

    Full Text Available Ground-penetrating radar (GPR is commonly used for locating burial sites. In this article, we acquired radargrams at a site where a domestic pig cadaver was buried. The measurements were conducted with the ProEx System GPR manufactured by the Swedish company Mala Geoscience with an antenna of 500MHz. The event corresponding to the pig can be clearly seen in the measurements. In order to improve the interpretation, the electromagnetic field is compared to numerical simulations computed with the pseudo-spectral Fourier method. A geological model has been defined on the basis of assumed electromagnetic properties (permittivity, conductivity and magnetic permeability. The results, when compared with the GPR measurements, show a dissimilar amplitude behaviour, with a stronger reflection event from the bottom of the pit. We have therefore performed another simulation by decreasing the electrical conductivity of the body very close to that of air. The comparison improved, showing more reflections, which could be an indication that the body contains air or has been degraded to a certain extent that the electrical resistivity has greatly increased.

  6. Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

    Directory of Open Access Journals (Sweden)

    El Andaloussi Samir

    2011-05-01

    Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

  7. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models

    NARCIS (Netherlands)

    Huang, Yunhong; Skwarek-Maruszewska, Aneta; Horré, Katrien; Vandewyer, Elke; Wolfs, Leen; Snellinx, An; Saito, Takashi; Radaelli, Enrico; Corthout, Nikky; Colombelli, Julien; Lo, Adrian C; Van Aerschot, Leen; Callaerts-Vegh, Zsuzsanna; Trabzuni, Daniah; Bossers, Koen; Verhaagen, Joost; Ryten, Mina; Munck, Sebastian; D'Hooge, Rudi; Swaab, Dick F; Hardy, John; Saido, Takaomi C; De Strooper, Bart; Thathiah, Amantha

    2015-01-01

    The orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer's disease (AD). However, given the vast

  8. Baicalein has protective effects on the 17β-estradiol-induced transformation of breast epithelial cells.

    Science.gov (United States)

    Chen, Yan; Wang, Jing; Hong, Duan-Yang; Chen, Lin; Zhang, Yan-Yan; Xu, Yi-Ni; Pan, Di; Fu, Ling-Yun; Tao, Ling; Luo, Hong; Shen, Xiang-Chun

    2017-02-07

    Epidemiologic and systematic studies have indicated that flavonoid consumption is associated with a lower incidence of breast cancer. Baicalein is the primary flavonoid derived from the roots of Scutellaria baicalensis Georgi. In the current study, the long-term exposure of breast epithelial cells to 17β-estradiol (E2) was used to investigate the chemopreventive potential of baicalein on neoplastic transformation. The results demonstrated that baicalein significantly inhibited E2-induced cell growth, motility, and invasiveness, and suppressed E2-induced misshapen acini formation in 3D cultures. Furthermore, it inhibited the ability of E2-induced cells to form clones in agarose and tumors in NOD/SCID immunodeficient mice. Docking studies using Sybyl-X 1.2 software showed that baicalein could bind to both estrogen receptor-α (ERa) and G-protein coupled estrogen receptor 30 (GPR30), which are two critical E2-mediated pathways. Baicalein prevented the E2-induced ERa-mediated activation of nuclear transcriptional signaling by interfering with the trafficking of ERa into the nucleus and subsequent binding to estrogen response elements, thereby decreasing the mRNA levels of ERa target genes. It also inhibited E2-induced GPR30-mediated signal transduction, as well as the transcription of GPR30-regulated genes. Therefore, these results suggest that baicalein is a potential drug for reducing the risk of estrogen-dependent breast cancer.

  9. Low resistance splices for HTS devices and applications

    Science.gov (United States)

    Lalitha, S. L.

    2017-09-01

    This paper discusses the preparation methodology and performance evaluation of low resistance splices made of the second generation (2G) high-temperature superconductor (HTS). These splices are required in a broad spectrum of HTS devices including a large aperture, high-field solenoid built in the laboratory to demonstrate a superconducting magnetic energy storage (SMES) device. Several pancake coils are assembled in the form of a nested solenoid, and each coil requires a hundred meters or more of 2G (RE)BCO tape. However, commercial availability of this superconductor with a very uniform physical properties is currently limited to shorter piece lengths. This necessitates us having splices to inter-connect the tape pieces within a pancake coil, between adjacent pancake coils, and to attach HTS current leads to the magnet assembly. As a part of the optimization and qualification of splicing process, a systematic study was undertaken to analyze the electrical performance of splices in two different configurations suitable for this magnet assembly: lap joint and spiral joint. The electrical performance is quantified in terms of the resistance of splices estimated from the current-voltage characteristics. It has been demonstrated that a careful application of this splicing technique can generate lap joints with resistance less than 1 nΩ at 77 K.

  10. GPR101 orphan GPCR: a novel cause of growth hormone deregulation

    OpenAIRE

    Abboud, Dayana; Daly, Adrian; Dupuis, Nadine; Laschet, Céline; Geubelle, Pierre; Pirotte, Bernard; BECKERS, Albert; Hanson, Julien

    2017-01-01

    GPR101 is an orphan G-protein coupled receptor with unknown ligand. In 2014, an international study clearly pointed to a strong association between this receptor and the X-linked acrogigantism (X-LAG) syndrome, which begins in childhood and causes the “tallest giants”. The children (carriers of the GPR101 duplication on the X chromosome) grow abnormally even before they are one year old, secrete phenomenal quantities of growth hormone, and develop pituitary adenomas that do not respond to cur...

  11. RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

    KAUST Repository

    Lee, Keh Chien

    2017-04-11

    The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3\\' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-β transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

  12. Alternative splicing of mutually exclusive exons--a review.

    Science.gov (United States)

    Pohl, Martin; Bortfeldt, Ralf H; Grützmann, Konrad; Schuster, Stefan

    2013-10-01

    Alternative splicing (AS) of pre-mRNAs in higher eukaryotes and several viruses is one major source of protein diversity. Usually, the following major subtypes of AS are distinguished: exon skipping, intron retention, and alternative 3' and 5' splice sites. Moreover, mutually exclusive exons (MXEs) represent a rare subtype. In the splicing of MXEs, two (or more) splicing events are not independent anymore, but are executed or disabled in a coordinated manner. In this review, several bioinformatics approaches for analyzing MXEs are presented and discussed. In particular, we revisit suitable definitions and nomenclatures, and bioinformatics tools for finding MXEs, adjacent and non-adjacent MXEs, clustered and grouped MXEs. Moreover, the molecular mechanisms for splicing MXEs proposed in the literature are reviewed and discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. [Identification of a novel GPR143 mutation in a Chinese family affected with X-linked ocular albinism].

    Science.gov (United States)

    Zhao, Qi; Guan, Menglong; Wang, Ling; Liao, Yong; Li-Ling, Jesse; Wan, Huajing

    2017-04-10

    To detect mutation of GPR143 gene in a Chinese patient affected with ocular albinism. Peripheral blood samples were collected from the proband and his parents. The coding regions of the GPR143 gene were subjected to PCR amplification and Sanger sequencing. A previously unreported mutation (c.758T>A) was found in exon 6 of the GPR143 gene in the proband and his mother. The same mutation was not found in his father. As predicted, the mutation has resulted in a stop codon, causing premature termination of protein translation. A novel mutation of the GPR143 gene related to X-linked ocular albinism has been identified.

  14. Test site experiments with a reconfigurable stepped frequency GPR

    Science.gov (United States)

    Persico, Raffaele; Matera, Loredana; Piro, Salvatore; Rizzo, Enzo; Capozzoli, Luigi

    2016-04-01

    In this contribution, some new possibilities offered by a reconfigurable stepped frequency GPR system are exposed. In particular, results achieved from a prototypal system achieved in two scientific test sites will be shown together with the results achieved in the same test sites with traditional systems. Moreover a novel technique for the rejection of undesired interferences is shown, with the use of interferences caused on purpose. Key words GPR, reconfigurable stepped frequency. Introduction A reconfigurable GPR system is meant as a GPR where some parameter can be changed vs. the frequency (if the system is stepped frequency) or vs. the time (if the system is pulsed) in a programmable way. The programming should then account for the conditions met in the scenario at hand [1]. Within the research project AITECH (http://www.aitechnet.com/ibam.html), the Institute for Archaeological and Monumental Heritage, together with the University of Florence and the IDS corporation have implemented a prototype, that has been used in sites of cultural interest in Italy [2], but also abroad in Norway and Malta. The system is a stepped frequency GPR working in the frequency range 50-1000 MHz, and its reconfigurability consists in three properties. The first one is the fact that the length of the antennas can be modulated by the aperture and closure of two electronic switches present along the arms of the antennas, so that the antennas can become electrically (and electronically) longer or shorter, so becoming more suitable to radiate some frequencies rather than some other. In particular, the system can radiate three different bands in the comprehensive range between 50-1000 MHz, so being suitable for different depth range of the buried targets, and the three bands are gathered in a unique "going through" because for each measurement point the system can sweep the entire frequency range trhee times, one for each configuration of the switchres on the arms. The second property is

  15. Revealing the Determinants of Widespread Alternative Splicing Perturbation in Cancer

    Directory of Open Access Journals (Sweden)

    Yongsheng Li

    2017-10-01

    Full Text Available It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine.

  16. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

    Directory of Open Access Journals (Sweden)

    Erik van der Wal

    2017-06-01

    Full Text Available The most common variant causing Pompe disease is c.-32-13T>G (IVS1 in the acid α-glucosidase (GAA gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

  17. Alternative Splicing of G9a Regulates Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Ana Fiszbein

    2016-03-01

    Full Text Available Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10 through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10+ isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  18. GPR and GPS data integration: examples of application in Antarctica

    Directory of Open Access Journals (Sweden)

    S. Gandolfi

    2001-06-01

    Full Text Available Ground Penetrating Radar (GPR and Global Positioning System (GPS techniques were employed in snow accumulation studies during the Italian leg of the International Trans-Antarctic Scientific Expedition (ITASE. The acquired data were useful both for glaciological and climatological studies. This paper presents some results obtained by GPR and GPS data integration employed to determine accumulation/ablation processes along the profile of the traverse that show how the snow-sublayer thickness can vary quickly in just a few kilometres. Some examples of data integration employed in detection and characterisation of buried crevasses are also presented.

  19. Minimising street work disruption by mapping cavities derived from 3D GPR-data: a new sewerage project in Torrente (Valencia, Spain)

    Science.gov (United States)

    Valls, Ana; Garcia, Francisco; Ramirez, Manuel; Benlloch, Javier

    2015-04-01

    Ground penetrating radar is usually employed for non-destructive detection of cavities in karst areas and road maintenance. This paper describes the inspection for cavity detection in a street located in Torrente (Valencia, Spain) where a new sewerage project was planned. Torrente population growth (more than 80,000 inhabitants last year) has caused urban development southwards from its downtown. According to municipality geologic configuration, new urbanized areas are located in mountains composed of limestone with presence of karst systems. During excavation work for a sewerage system installation, a 4 x 2 x 1.5 m shallow cave was found in one planned street. For this reason, digging activities were stopped and a GPR survey was carried out on the street. A 1x1 m grid was collected using a GSSI SIR-3000 equipment. A 400 MHz frequency antenna was used for reaching 2.5 m approx. depth, attending the characteristics of the discovered cave and the excavation project depth. GPR records were calibrated in situ, thanks to the unearthed cavity. The 3D GPR-data interpretation mapped several caves only on one side of the street. The detected cavities coincided with the sewerage system layout. These underground spaces were isolated from each other, as small individual karst caves. The outcomes of this study allowed the modification of the sewerage project. Therefore, the sewerage system layout was moved to the other side of the street where no cavities were detected with the GPR survey. GPR is proved to be an efficient tool to be taken into consideration by civil engineers and architects for designing new infrastructures (e.g. sewerage systems) in urban planning areas. We conclude GPR helps minimising cost, time and inconveniences to neighbourhood during excavation works, especially in cities.

  20. Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183)

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Norn, Christoffer; Laurent, Stephane

    2012-01-01

    , the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about...

  1. GPR56-Related Polymicrogyria: Clinicoradiologic Profile of 4 Patients.

    Science.gov (United States)

    Desai, Neelu A; Udani, Vrajesh

    2015-11-01

    Bilateral frontoparietal polymicrogyria is an autosomal recessive cortical malformation associated with abnormalities of neuronal migration, white matter changes, and mild brainstem and cerebellar abnormalities. Affected patients present with delayed milestones, intellectual disability, epilepsy, ataxia, and eye movement abnormalities. The clinicoradiologic profile resembles congenital muscular dystrophy. However, no muscle disease or characteristic eye abnormalities of congenial muscular dystrophy are detected in these children. GPR56 is the only confirmed gene associated with bilateral frontoparietal polymicrogyria. Antenatal diagnosis is possible if the index case is genetically confirmed. Four patients from different Indian families with a distinct clinicoradiologic profile resembling congenital muscular dystrophy with mutations in the GPR56 gene are described. © The Author(s) 2015.

  2. Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents.

    Directory of Open Access Journals (Sweden)

    Ola Fjellström

    Full Text Available Type 2 diabetes (T2D occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.

  3. The peculiarities of large intron splicing in animals.

    Directory of Open Access Journals (Sweden)

    Samuel Shepard

    Full Text Available In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These "large introns" must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5' and 3' acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing-a consecutive splicing from the 5'-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites.

  4. Systematic Analysis of Splice-Site-Creating Mutations in Cancer

    Directory of Open Access Journals (Sweden)

    Reyka G. Jayasinghe

    2018-04-01

    Full Text Available Summary: For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. : Jayasinghe et al. identify nearly 2,000 splice-site-creating mutations (SCMs from over 8,000 tumor samples across 33 cancer types. They provide a more accurate interpretation of previously mis-annotated mutations, highlighting the importance of integrating data types to understand the functional and the clinical implications of splicing mutations in human disease. Keywords: splicing, RNA, mutations of clinical relevance

  5. PathwaySplice: An R package for unbiased pathway analysis of alternative splicing in RNA-Seq data.

    Science.gov (United States)

    Yan, Aimin; Ban, Yuguang; Gao, Zhen; Chen, Xi; Wang, Lily

    2018-04-24

    Pathway analysis of alternative splicing would be biased without accounting for the different number of exons or junctions associated with each gene, because genes with higher number of exons or junctions are more likely to be included in the "significant" gene list in alternative splicing. We present PathwaySplice, an R package that (1) Performs pathway analysis that explicitly adjusts for the number of exons or junctions associated with each gene; (2) Visualizes selection bias due to different number of exons or junctions for each gene and formally tests for presence of bias using logistic regression; (3) Supports gene sets based on the Gene Ontology terms, as well as more broadly defined gene sets (e.g. MSigDB) or user defined gene sets; (4) Identifies the significant genes driving pathway significance and (5) Organizes significant pathways with an enrichment map, where pathways with large number of overlapping genes are grouped together in a network graph. https://bioconductor.org/packages/release/bioc/html/PathwaySplice.html. lily.wangg@gmail.com, xi.steven.chen@gmail.com.

  6. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitat...... describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3' splice site (3'ss).......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

  7. GPR Raw-Data Order Statistic Filtering and Split-Spectrum Processing to Detect Moisture

    Directory of Open Access Journals (Sweden)

    Gokhan Kilic

    2014-05-01

    Full Text Available Considerable research into the area of bridge health monitoring has been undertaken; however, information is still lacking on the effects of certain defects, such as moisture ingress, on the results of ground penetrating radar (GPR surveying. In this paper, this issue will be addressed by examining the results of a GPR bridge survey, specifically the effect of moisture in the predicted position of the rebars. It was found that moisture ingress alters the radargram to indicate distortion or skewing of the steel reinforcements, when in fact destructive testing was able to confirm that no such distortion or skewing had occurred. Additionally, split-spectrum processing with order statistic filters was utilized to detect moisture ingress from the GPR raw data.

  8. Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

    Science.gov (United States)

    Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

    2008-06-01

    Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

  9. Topographic Correction of GPR Profiles Based on Laser Data

    International Nuclear Information System (INIS)

    Zhang, Di; Zhong, Ruofei; Li, Jia Cun; Zeng, Fanyang

    2014-01-01

    Data obtained by GPR (Ground Penetrating Radar) are displayed as a continuous cross-sectional profile. Surface, generally, is not flat. As a result, the image becomes distorted and the depth calculated from the surface no longer represents the true and exact position of electrically distinctive layers and objects in materials. In order to get real geologic cross section, GPR data must be corrected. This is paper discusses a new method using the color point cloud data obtained by a Vehicle-borne laser scanning system to compensate for elevation fluctuate. Elevation profile can be extracted from topographic data of survey site acquired using laser scanner, which can then be used to offset the error of GPR data. Through the discrete points in the survey line, each trace of the profile has its own elevation value showing a vertical difference from the reference profile with maximum elevation, then time shifts value of traces vertical offset versus the reference trace of profile can be obtained. At last, the results of topographic correction for radargrams that look extremely like the real geologic cross section are presented, which allows us to get a better profile interpretation and position of the objects and layers in the subsurface

  10. Disposal of iron tailings in reservoirs: a GPR application

    Directory of Open Access Journals (Sweden)

    Rachel Jardim Martini

    2016-11-01

    Full Text Available A large volume of waste is generated by iron mining in Brazil, and the amount has been rapidly increasing. The waste is usually stored in large piles or in reservoirs formed by tailings dams, which occupy large areas in the mining complex. The limitation of natural resources and of new areas for waste disposal has led to change in paradigms. This study therefore aimed to apply a geophysical technique, known as Ground Penetrating Radar (GPR, in order to define sedimentation patterns in the subsurface reservoir created by Diogo's tailings dam in Rio Piracicaba in Minas Gerais, Brazil. To assist the recognition of patterns captured by the GPR, subsurface material samples were collected and analyzed for mineralogical composition, moisture content, electrical conductivity, mineralogical analysis, particle size, X-Ray Diffraction and X-Ray Fluorescence. The results indicated that areas with tailings that had high concentrations of hematite (around 60% in the mineralogical analysis altered the reflection patterns. The presence of water generated some multiple reflections, which were less significant in shallower sites with more waste and more significant in deeper sites with less waste. In general, the application of the GPR was feasible in aquatic environments with rich subsurface hematite deposits.

  11. Experimental results from a stepped frequency GPR

    Directory of Open Access Journals (Sweden)

    G. M. Signore

    2003-06-01

    Full Text Available In the framework of a nationally funded project, a Ground Penetrating Radar (GPR has been developed by the Italian Consortium for Research on Advanced Remote Sensing Systems (CO.RI.S.T.A.. The system was described in a previous paper (Alberti et al., 2002. As new aspects, the system is a stepped frequency GPR that can work both in gated and ungated mode, and the antennas can be moved automatically in a controlled fashion. As aspects of geophysical interest, the system is exploitable in situations wherein a high resolution and a shallow penetration in the soil (a few meters are required. Possibly, this is an example of probing a landscape. This paper completes the results of Alberti et al. (2002, wherein laboratory tests where described, by providing the main results obtained during an outdoor experimental campaign, performed fi rst in a controlled site and then in an archaeological site.

  12. Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich diets.

    Science.gov (United States)

    Du Toit, Eugene; Browne, Liam; Irving-Rodgers, Helen; Massa, Helen M; Fozzard, Nicolette; Jennings, Michael P; Peak, Ian R

    2017-04-20

    Although there is good evidence showing that diets rich in medium chain fatty acids (MCFAs) have less marked obesogenic and diabetogenic effects than diets rich in long chain fatty acids (LCFAs), the role of the pro-inflammatory, medium chain fatty acid receptor (GPR84) in the aetiology of obesity and glucose intolerance is not well characterised. We set out to determine whether GPR84 expression influences obesity and glucose intolerance susceptibility in MCFA and LCFA rich diet fed mice. Wild type (WT) and GPR84 knockout (KO) mice were fed a control, MCFA or LCFA diet, and body mass, heart, liver and epididymal fat mass was assessed, as well as glucose tolerance and adipocyte size. LCFA diets increased body mass and decreased glucose tolerance in both WT and GPR84 KO animals while MCFA diets had no effect on these parameters. There were no differences in body weight when comparing WT and GPR84 KO mice on the respective diets. Glucose tolerance was also similar in WT and GPR84 KO mice irrespective of diet. Liver mass was increased following LCFA feeding in WT but not GPR84 KO mice. Hepatic triglyceride content was increased in GPR84 KO animals fed MCFA, and myocardial triglyceride content was increased in GPR84 KO animals fed LCFA. GPR84 deletion had no effects on body weight or glucose tolerance in mice fed either a high MCFA or LCFA diet. GPR84 may influence lipid metabolism, as GPR84 KO mice had smaller livers and increased myocardial triglyceride accumulation when fed LCFA diets, and increased liver triglyceride accumulation in responses to increased dietary MCFAs.

  13. LH-21 and abnormal cannabidiol improve β-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

    Science.gov (United States)

    Ruz-Maldonado, Inmaculada; Pingitore, Attilio; Liu, Bo; Atanes, Patricio; Huang, Guo Cai; Baker, David; Alonso, Francisco José; Bermúdez-Silva, Francisco Javier; Persaud, Shanta J

    2018-04-01

    To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55 -/- mice. Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca 2+ ] i, cAMP , apoptosis, β-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca 2+ ] i in human islets and islets from both GPR55 +/+ and GPR55 -/- mice. LH-21 also increased insulin secretion and [Ca 2+ ] i in human islets and GPR55 +/+ mouse islets, but concentrations of LH-21 up to 0.1 μM were ineffective in islets from GPR55 -/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55 +/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased β-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55 -/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. This study showed that Abn-CBD and LH-21 improve human and mouse islet β-cell function and viability. Use of islets from GPR55 -/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised. © 2017 John Wiley & Sons Ltd.

  14. Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing.

    Science.gov (United States)

    Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu; Marangoudakis, Spiro; Lipscombe, Diane

    2010-10-01

    Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances μ-opioid receptor-mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.

  15. Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events.

    Science.gov (United States)

    Baudhuin, Linnea M; Kotzer, Katrina E; Lagerstedt, Susan A

    2015-03-01

    Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype-phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome. Genotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed. A higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria-positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P Marfan syndrome patients with FBN1 truncating and splicing variants.Genet Med 17 3, 177-187.

  16. Features generated for computational splice-site prediction correspond to functional elements

    Directory of Open Access Journals (Sweden)

    Wilbur W John

    2007-10-01

    Full Text Available Abstract Background Accurate selection of splice sites during the splicing of precursors to messenger RNA requires both relatively well-characterized signals at the splice sites and auxiliary signals in the adjacent exons and introns. We previously described a feature generation algorithm (FGA that is capable of achieving high classification accuracy on human 3' splice sites. In this paper, we extend the splice-site prediction to 5' splice sites and explore the generated features for biologically meaningful splicing signals. Results We present examples from the observed features that correspond to known signals, both core signals (including the branch site and pyrimidine tract and auxiliary signals (including GGG triplets and exon splicing enhancers. We present evidence that features identified by FGA include splicing signals not found by other methods. Conclusion Our generated features capture known biological signals in the expected sequence interval flanking splice sites. The method can be easily applied to other species and to similar classification problems, such as tissue-specific regulatory elements, polyadenylation sites, promoters, etc.

  17. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes

    Directory of Open Access Journals (Sweden)

    Elvezia Maria Paraboschi

    2015-09-01

    Full Text Available Abnormalities in RNA metabolism and alternative splicing (AS are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls, followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p = 0.0015 by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

  18. Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Bang, Jeanne Mv; Christensen, Lise L

    2018-01-01

    designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific...

  19. Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

    Science.gov (United States)

    van der Klift, Heleen M; Jansen, Anne M L; van der Steenstraten, Niki; Bik, Elsa C; Tops, Carli M J; Devilee, Peter; Wijnen, Juul T

    2015-01-01

    A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants. PMID:26247049

  20. Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Aman C. Kaushik

    2018-02-01

    Full Text Available GPR142 (G protein receptor 142 is a novel orphan GPCR (G protein coupled receptor belonging to “Class A” of GPCR family and expressed in β cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling, and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

  1. Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

    Science.gov (United States)

    Kaushik, Aman C.; Kumar, Sanjay; Wei, Dong Q.; Sahi, Shakti

    2018-02-01

    GPR142 (G protein receptor 142) is a novel orphan GPCR (G protein coupled receptor) belonging to ‘Class A’ of GPCR family and expressed in beta cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

  2. Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs.

    Science.gov (United States)

    Miyabe, Masahiro; Gin, Azusa; Onozawa, Eri; Daimon, Mana; Yamada, Hana; Oda, Hitomi; Mori, Akihiro; Momota, Yutaka; Azakami, Daigo; Yamamoto, Ichiro; Mochizuki, Mariko; Sako, Toshinori; Tamura, Katsutoshi; Ishioka, Katsumi

    2015-10-01

    G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4-5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.

  3. Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

    Science.gov (United States)

    Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

    2017-08-01

    Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. CD36- and GPR120-mediated Ca²⁺ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice.

    Science.gov (United States)

    Ozdener, Mehmet Hakan; Subramaniam, Selvakumar; Sundaresan, Sinju; Sery, Omar; Hashimoto, Toshihiro; Asakawa, Yoshinori; Besnard, Philippe; Abumrad, Nada A; Khan, Naim Akhtar

    2014-04-01

    It is important to increase our understanding of gustatory detection of dietary fat and its contribution to fat preference. We studied the roles of the fat taste receptors CD36 and GPR120 and their interactions via Ca(2+) signaling in fungiform taste bud cells (TBC). We measured Ca(2+) signaling in human TBC, transfected with small interfering RNAs against messenger RNAs encoding CD36 and GPR120 (or control small interfering RNAs). We also studied Ca(2+) signaling in TBC from CD36(-/-) mice and from wild-type lean and obese mice. Additional studies were conducted with mouse enteroendocrine cell line STC-1 that express GPR120 and stably transfected with human CD36. We measured release of serotonin and glucagon-like peptide-1 from human and mice TBC in response to CD36 and GPR120 activation. High concentrations of linoleic acid induced Ca(2+) signaling via CD36 and GPR120 in human and mice TBC, as well as in STC-1 cells, and low concentrations induced Ca(2+) signaling via only CD36. Incubation of human and mice fungiform TBC with lineoleic acid down-regulated CD36 and up-regulated GPR120 in membrane lipid rafts. Obese mice had decreased spontaneous preference for fat. Fungiform TBC from obese mice had reduced Ca(2+) and serotonin responses, but increased release of glucagon-like peptide-1, along with reduced levels of CD36 and increased levels of GPR120 in lipid rafts. CD36 and GPR120 have nonoverlapping roles in TBC signaling during orogustatory perception of dietary lipids; these are differentially regulated by obesity. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  5. Full 3D Microwave Tomography enhanced GPR surveys: a case study

    Science.gov (United States)

    Catapano, Ilaria; Soldovieri, Francesco; Affinito, Antonio; Hugenschmidt, Johannes

    2014-05-01

    Ground Penetrating Radar (GPR) systems are well assessed non-invasive diagnostic tools capable of providing high resolution images of the inner structure of the probed spatial region. Owing to this capability, GPR systems are nowadays more and more considered in the frame of civil engineering surveys since they may give information on constructive details as well as on the aging and risk factors affecting the healthiness of an infrastructure. In this frame, accurate, reliable and easily interpretable images of the probed scenarios are mandatory in order to support the management of maintenance works and assure the safety of structures. Such a requirement motivates the use of different and sophisticated data processing approaches in order to compare more than one image of the same scene, thus improving the reliability and objectiveness of the GPR survey results. Among GPR data processing procedures, Microwave Tomography approaches based on the Born approximation face the imaging as the solution of a linear inverse problem, which is solved by using the Truncated Singular Value Decomposition as a regularized inversion scheme [1]. So far, an approach exploiting a 2D scalar model of the scattering phenomenon have been adopted to process GPR data gathered along a single scan. In this case, 3D images are obtained by interpolating 2D reconstructions (this is referred commonly as pseudo 3D imaging). Such an imaging approach have provided valuable results in several real cases dealing with not only surveys for civil engineering but also archeological prospection, subservice monitoring, security surveys and so on [1-4]. These encouraging results have motivated the development of a full 3D Microwave Tomography approach capable of accounting for the vectorial nature of the wave propagation. The reconstruction capabilities of this novel approach have been assessed mainly against experimental data collected in laboratory controlled conditions. The obtained results corroborate

  6. Microwave tomography for an effective imaging in GPR on UAV/airborne observational platforms

    Science.gov (United States)

    Soldovieri, Francesco; Catapano, Ilaria; Ludeno, Giovanni

    2017-04-01

    GPR was originally thought as a non-invasive diagnostics technique working in contact with the underground or structure to be investigated. On the other hand, in the recent years several challenging necessities and opportunities entail the necessity to work with antenna not in contact with the structure to be investigated. This necessity arises for example in the case of landmine detection but also for cultural heritage diagnostics. Other field of application regards the forward-looking GPR aiming at shallower hidden targets forward the platfrom (vehicle) carrying the GPR [1]. Finally, a recent application is concerned with the deployment of airborne/UAV GPR, able to ensure several advantages in terms of large scale surveys and "freedom" of logistics constraint [2]. For all the above mentioned cases, the interest is towards the development of effective data processing able to make imaging task in real time. The presentation will show different data processing strategies, based on microwave tomography [1,2], for a reliable and real time imaging in the case of GPR platforms far from the interface of the structure/underground to be investigated. [1] I. Catapano, A. Affinito, A. Del Moro,.G. Alli, and F. Soldovieri, "Forward-Looking Ground-Penetrating Radar via a Linear Inverse Scattering Approach," IEEE Transactions on Geoscience and Remote Sensing, vol. 53, pp. 5624 - 5633, Oct. 2015. [2] I. Catapano, L. Crocco, Y. Krellmann, G. Triltzsch, and F. Soldovieri, "A tomographic approach for helicopter-borne ground penetrating radar imaging," IEEE Geosci. Remote Sens. Lett., vol. 9, no. 3, pp. 378-382, May 2012.

  7. The splicing of tiny introns of Paramecium is controlled by MAGO.

    Science.gov (United States)

    Contreras, Julia; Begley, Victoria; Marsella, Laura; Villalobo, Eduardo

    2018-07-15

    The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Neural network based inspection of voids and karst conduits in hydro-electric power station tunnels using GPR

    Science.gov (United States)

    Kilic, Gokhan; Eren, Levent

    2018-04-01

    This paper reports on the fundamental role played by Ground Penetrating Radar (GPR), alongside advanced processing and presentation methods, during the tunnel boring project at a Dam and Hydro-Electric Power Station. It identifies from collected GPR data such issues as incomplete grouting and the presence of karst conduits and voids and provides full details of the procedures adopted. In particular, the application of collected GPR data to the Neural Network (NN) method is discussed.

  9. pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

    Science.gov (United States)

    Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

    2011-01-01

    Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

  10. Safety in GPR prospecting: a rarely-considered issue

    Science.gov (United States)

    Persico, Raffaele; Pajewski, Lara; Trela, Christiane; Carrick Utsi, Erica

    2016-04-01

    Safety issues (of people first of all, but also of the equipment and environment) are rarely considered in Ground-Penetrating Radar (GPR) prospecting and, more in general, in near-surface geophysical prospecting. As is right and fully understandable, the scientific community devotes greatest attention first of all to the theoretical and practical aspects of GPR technique, affecting the quality of attainable results, secondly to the efforts and costs needed to achieve them [1-2]. However, the (luckily) growing GPR market and range of applications make it worth giving serious consideration to safety issues, too. The existing manuals dealing with safety in geophysics are mainly concerned with applications requiring "deep" geophysical prospecting, for example the search for oilfields and other hydrocarbon resources [3]. Near-surface geophysics involves less dangers than deep geophysics, of course. Nevertheless, several accidents have already happened during GPR experimental campaigns. We have personally had critical experiences and collected reliable testimonies concerning occurred problems as mountain sicks, fractures of legs, stomach problems, allergic reactions, encounters with potentially-dangerous animals, and more. We have also noticed that much more attention is usually paid to safety issues during indoor experimental activities (in laboratory), rather than during outdoor fieldworks. For example, the Italian National research Council is conventioned with safety experts who hold periodical seminaries about safety aspects. Having taken part to some of them, to our experience we have never heard a "lecture" devoted to outdoor prospecting. Nowadays, any aspects associated to the use of the technologies should be considered. The increasing sensibility and sense of responsibility towards environmental matters impose GPR end-users to be careful not to damage the environment and also the cultural heritage. Near-surface prospecting should not compromise the flora and

  11. GPR143 gene mutation analysis in pediatric patients with albinism.

    Science.gov (United States)

    Trebušak Podkrajšek, Katarina; Stirn Kranjc, Branka; Hovnik, Tinka; Kovač, Jernej; Battelino, Tadej

    2012-09-01

    X-linked ocular albinism type 1 is difficult to differentiate clinically from other forms of albinism in young patients. X-linked ocular albinism type 1 is caused by mutations in the GPR143 gene, encoding melanosome specific G-protein coupled receptor. Patients typically present with moderately to severely reduced visual acuity, nystagmus, strabismus, photophobia, iris translucency, hypopigmentation of the retina, foveal hypoplasia and misrouting of optic nerve fibers at the chiasm. Following clinical ophthalmological evaluation, GPR143 gene mutational analyses were performed in a cohort of 15 pediatric male patients with clinical signs of albinism. Three different mutations in the GPR143 gene were identified in four patients, including a novel c.886G>A (p.Gly296Arg) mutation occurring "de novo" and a novel intronic c.360 + 5G>A mutation, identified in two related boys. Four patients with X-linked ocular albinism type 1 were identified from a cohort of 15 boys with clinical signs of albinism using mutation detection methods. Genetic analysis offers the possibility of early definitive diagnosis of ocular albinism type 1 in a significant portion of boys with clinical signs of albinism.

  12. Crevasse detection with GPR across the Ross Ice Shelf, Antarctica

    Science.gov (United States)

    Delaney, A.; Arcone, S.

    2005-12-01

    We have used 400-MHz ground penetrating radar (GPR) to detect crevasses within a shear zone on the Ross Ice Shelf, Antarctica, to support traverse operations. The transducer was attached to a 6.5-m boom and pushed ahead of an enclosed tracked vehicle. Profile speeds of 4.8-11.3 km / hr allowed real-time crevasse image display and a quick, safe stop when required. Thirty-two crevasses were located with radar along the 4.8 km crossing. Generally, crevasse radar images were characterized by dipping reflections above the voids, high-amplitude reflections originating from ice layers at the base of the snow-bridges, and slanting, diffracting reflections from near-vertical crevasse walls. New cracks and narrow crevasses (back-filling with bulldozed snow, afforded an opportunity to ground-truth GPR interpretations by comparing void size and snow-bridge geometry with the radar images. While second and third season radar profiles collected along the identical flagged route confirmed stability of the filled crevasses, those profiles also identified several new cracks opened by ice extension. Our experiments demonstrate capability of high-frequency GPR in a cold-snow environment for both defining snow layers and locating voids.

  13. Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Liu, H X; Goodall, G J; Kole, R; Filipowicz, W

    1995-01-16

    We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.

  14. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

  15. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  16. GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer's disease.

    Science.gov (Unit