Sample records for targeting myeloperoxidase identifies

  1. Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase*

    PubMed Central

    Chapman, Anna L. P.; Mocatta, Tessa J.; Shiva, Sruti; Seidel, Antonia; Chen, Brian; Khalilova, Irada; Paumann-Page, Martina E.; Jameson, Guy N. L.; Winterbourn, Christine C.; Kettle, Anthony J.

    2013-01-01

    Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the FeV redox intermediate of myeloperoxidase, to Compound II, which has FeIV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation. PMID:23306200

  2. Genetic variants influencing elevated myeloperoxidase levels increase risk of stroke.

    PubMed

    Phuah, Chia-Ling; Dave, Tushar; Malik, Rainer; Raffeld, Miriam R; Ayres, Alison M; Goldstein, Joshua N; Viswanathan, Anand; Greenberg, Steven M; Jagiella, Jeremiasz M; Hansen, Björn M; Norrving, Bo; Jimenez-Conde, Jordi; Roquer, Jaume; Pichler, Alexander; Enzinger, Christian; Montaner, Joan; Fernandez-Cadenas, Israel; Lindgren, Arne; Slowik, Agnieszka; Schmidt, Reinhold; Biffi, Alessandro; Rost, Natalia; Langefeld, Carl D; Markus, Hugh S; Mitchell, Braxton D; Worrall, Brad B; Kittner, Steven J; Woo, Daniel; Dichgans, Martin; Rosand, Jonathan; Anderson, Christopher D

    2017-10-01

    Primary intracerebral haemorrhage and lacunar ischaemic stroke are acute manifestations of progressive cerebral microvascular disease. Current paradigms suggest atherosclerosis is a chronic, dynamic, inflammatory condition precipitated in response to endothelial injury from various environmental challenges. Myeloperoxidase plays a central role in initiation and progression of vascular inflammation, but prior studies linking myeloperoxidase with stroke risk have been inconclusive. We hypothesized that genetic determinants of myeloperoxidase levels influence the development of vascular instability, leading to increased primary intracerebral haemorrhage and lacunar stroke risk. We used a discovery cohort of 1409 primary intracerebral haemorrhage cases and 1624 controls from three studies, an extension cohort of 12 577 ischaemic stroke cases and 25 643 controls from NINDS-SiGN, and a validation cohort of 10 307 ischaemic stroke cases and 29 326 controls from METASTROKE Consortium with genome-wide genotyping to test this hypothesis. A genetic risk score reflecting elevated myeloperoxidase levels was constructed from 15 common single nucleotide polymorphisms identified from prior genome-wide studies of circulating myeloperoxidase levels (P < 5 × 10-6). This genetic risk score was used as the independent variable in multivariable regression models for association with primary intracerebral haemorrhage and ischaemic stroke subtypes. We used fixed effects meta-analyses to pool estimates across studies. We also used Cox regression models in a prospective cohort of 174 primary intracerebral haemorrhage survivors for association with intracerebral haemorrhage recurrence. We present effects of myeloperoxidase elevating single nucleotide polymorphisms on stroke risk per risk allele, corresponding to a one allele increase in the myeloperoxidase increasing genetic risk score. Genetic determinants of elevated circulating myeloperoxidase levels were associated with both primary

  3. Immunomagnetic separation of human myeloperoxidase using an antibody-mimicking peptide identified by phage display.

    PubMed

    Yun, Soi; Ryu, Hyunmin; Lee, E K

    2017-09-10

    Phage display biopanning is a powerful in vitro selection process for screening and identifying peptides that bind to a target protein of interest. With the aim of replacing antibodies in immuno-diagnostic applications, we identified peptides whose binding characteristics mimicked those of anti-human myeloperoxidase (hMPO), a biomarker for acute cardiac diseases. Based on ELISA results from four phage clones, we selected and chemically synthesized a 12-mer peptide (SYIEPPERHRHR). Quartz crystal microbalance and surface plasmon resonance analyses revealed that the molar binding equilibrium ratio of the synthesized peptide was 0.023, approximately 43-fold lower than that of the anti-hMPO antibody. The dissociation constant (K d ) was 57nM, which was comparable to that of the native antibody (83nM). Next, we biotinylated the peptide at its N-terminus and attached the biotinylated peptide to the surface of streptavidin-coated magnetic particles to assess its ability to selectively capture hMPO. The binding equilibrium data were similar to the previous analyses; specifically, around 0.021mol peptide bound to 1mol of hMPO. Antigen capture was found to be selective and to be relatively little influenced by the presence of human serum albumin (HSA), an abundant constituent of serum. Our work demonstrates the potential of immunomagnetic isolation to achieve selective capture of a low-concentration antigen from complex solutions such as serum. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Enzyme-Sensitive MR Imaging Targeting Myeloperoxidase Identifies Active Inflammation in Experimental Rabbit Atherosclerotic Plaques

    PubMed Central

    Ronald, John A.; Chen, John W.; Chen, Yuanxin; Hamilton, Amanda M.; Rodriguez, Elisenda; Reynolds, Fred; Hegele, Robert A.; Rogers, Kem A.; Querol, Manel; Bogdanov, Alexei; Weissleder, Ralph; Rutt, Brian K.

    2009-01-01

    Background Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase (MPO) is a central inflammatory enzyme secreted by activated macrophages, and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance (MR) agent can visualize MPO activity in atherosclerotic plaques in a rabbit model. Methods and Results We performed MR imaging of the thoracic aorta of New Zealand white (NZW) rabbits fed a cholesterol (n=11) or normal (n=4) diet up to 2 hours after injection of the MPO sensor bis-5HT-DTPA(Gd) (MPO(Gd)), the conventional agent, DTPA(Gd), or an MPO (Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hour post injection) showed focal areas of increased contrast (>2-fold) in diseased wall, but not in normal wall (p=0.84), compared to both DTPA(Gd) (n=11; p<0.001) and bis-tyr-DTPA(Gd) (n=3; p<0.05). Biochemical assays confirmed that diseased wall possessed three-fold elevated MPO activity compared to normal wall (p<0.01). Areas detected by MPO(Gd) imaging co-localized and correlated with MPO-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, p<0.0001). While macrophages were the main source of MPO, not all macrophages secreted MPO, suggesting that distinct subpopulations contribute differently to atherogenesis and supporting our functional approach. Conclusions Our study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme, to noninvasively provide both anatomic and functional information in vivo. PMID:19652086

  5. Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System

    PubMed Central

    Klebanoff, Seymour J.

    1968-01-01

    An antibacterial effect of myeloperoxidase, a halide, such as iodide, bromide, or chloride ion, and H2O2 on Escherichia coli or Lactobacillus acidophilus is described. When L. acidophilus was employed, the addition of H2O2 was not required; however, the protective effect of catalase suggested that, in this instance, H2O2 was generated by the organisms. The antibacterial effect was largely prevented by preheating the myeloperoxidase at 80 C or greater for 10 min or by the addition of a number of inhibitors; it was most active at the most acid pH employed (5.0). Lactoperoxidase was considerably less effective than was myeloperoxidase when chloride was the halide employed. Myeloperoxidase, at high concentrations, exerted an antibacterial effect on L. acidophilus in the absence of added halide, which also was temperature- and catalase-sensitive. Peroxidase was extracted from intact guinea pig leukocytes by weak acid, and the extract with peroxidase activity had antibacterial properties which were similar, in many respects, to those of the purified preparation of myeloperoxidase. Under appropriate conditions, the antibacterial effect was increased by halides and by H2O2 and was decreased by catalase, as well as by cyanide, azide, Tapazole, and thiosulfate. This suggests that, under the conditions employed, the antibacterial properties of a weak acid extract of guinea pig leukocytes is due, in part, to its peroxidase content, particularly if a halide is present in the reaction mixture. A heat-stable antibacterial agent or agents also appear to be present in the extract. PMID:4970226

  6. Myeloperoxidase: a target for new drug development?

    PubMed Central

    Malle, E; Furtmüller, P G; Sattler, W; Obinger, C

    2007-01-01

    Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low- and high-spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers. PMID:17592500

  7. Isolation and molecular cloning of a fish myeloperoxidase.

    PubMed

    Castro, Rosario; Piazzon, M Carla; Noya, Manuel; Leiro, José M; Lamas, Jesús

    2008-01-01

    Myeloperoxidase (MPO) is a conspicuous enzyme in neutrophils of many fish species. Although the MPO gene has been identified in some fish species, the structure and functions of the protein remain to be determined in these vertebrates. In the present study, we isolated turbot neutrophil MPO from kidney cells by affinity chromatography, with Ulva rigida acidic sulphated polysaccharides (ASP), some of which resemble glycosaminoglycans, and Sepharose. The product obtained, of approximately 150kDa molecular weight and with peroxidase activity, was examined by SDS-page electrophoresis under reduced conditions and immunoblotting, and a single band of about 75kDa was observed. The results obtained suggest that turbot MPO is a dimer and that the band of 75kDa probably corresponds to a monomer generated by treatment of the samples with the reducing agent. The band was analysed by electromatrix-assisted laser desorption ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-ion trap mass spectrometry, dynamic exclusion mode (LC-ESI-IT DE), to determine the amino acid composition of some peptides. The peptides obtained were very similar to myeloperoxidases of other organisms, including other fish and mammals, and were used to design the primers for cDNA amplification. A 567bp product was amplified and the deduced amino acid sequence, which contains several putative N-glycosylation and O-glycosylation sites, was compared with other myeloperoxidases. As expected, turbot MPO was more similar to MPO from other fish species (67-86% identity), where the phylogenetic tree obtained agrees with the taxonomic hierarchy, than to MPO from mammals (55-57% identity) and other groups. The results obtained in the present study will also allow functional studies to be carried out with turbot neutrophil MPO enzyme, as well as analysis of MPO gene expression under different stimuli.

  8. Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation.

    PubMed

    Zgliczyński, J M; Stelmaszyńska, T; Olszowska, E; Krawczyk, A; Kwasnowska, E; Wróbel, J T

    1983-01-01

    It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.

  9. Relation of myeloperoxidase-463G/A polymorphism with metabolic syndrome and its component traits in Egyptian women.

    PubMed

    Mehanna, Eman T; Saleh, Samy M; Ghattas, Maivel H; Mesbah, Noha M; Abo-Elmatty, Dina M

    2015-02-01

    Myeloperoxidase is a heme protein secreted by activated macrophages and generates intermediates that oxidize lipoproteins. Myeloperoxidase-463G/A is a functional polymorphism involved in regulation of myeloperoxidase expression. The aim of this study is to assess the relation of myeloperoxidase-463G/A polymorphism with metabolic syndrome and its component traits in Egyptian women from the Suez Canal area. The study includes 100 healthy female subjects and 100 metabolic syndrome patients. The component traits of metabolic syndrome are determined and the genotypes of the polymorphisms assessed using the PCR-RFLP technique. There was no significant difference in the allele frequencies between the metabolic syndrome and control groups. However, the GA and AA genotypes were associated with lower total cholesterol, LDL-C, systolic and diastolic blood pressure in the patients. Myeloperoxidase-463G/A polymorphism is not associated with the incidence of metabolic syndrome.

  10. Myeloperoxidase can differentiate between sepsis and non-infectious SIRS and predicts mortality in intensive care patients with SIRS.

    PubMed

    Schrijver, Irene T; Kemperman, Hans; Roest, Mark; Kesecioglu, Jozef; de Lange, Dylan W

    2017-09-15

    Systemic inflammatory response syndrome (SIRS) is a clinical syndrome following inflammation. Clinically, it is difficult to distinguish SIRS following an infection, i.e., sepsis, from non-infectious SIRS. Myeloperoxidase is a hemeprotein stored in the neutrophil azurophilic granules and is one of the main pillars of neutrophil attack. Therefore, we hypothesized that myeloperoxidase can differentiate between sepsis and non-infectious SIRS in patients with systemic inflammatory response syndrome in the intensive care unit (ICU). An observational single-center cohort study was conducted measuring myeloperoxidase in patients with SIRS in the first 48 h after admission. The outcomes were established using predefined definitions. Thirty-day mortality was retrospectively assessed. We found significantly higher levels of myeloperoxidase in patients with sepsis and septic shock compared to patients without sepsis (60 ng/ml versus 43 ng/ml, P = 0.002). Myeloperoxidase levels were related to 30-day mortality (P = 0.032), and high MPO levels on top of a high APACHE IV score further increased mortality risk. We show that myeloperoxidase is a potentially novel biomarker for sepsis in the ICU. Myeloperoxidase could eventually help in diagnosing sepsis and predicting mortality. However, more research is necessary to confirm our results.

  11. The effect on serum myeloperoxidase activity and oxidative status of eradication treatment in patients Helicobacter pylori infected.

    PubMed

    Nazligul, Yaşar; Aslan, Mehmet; Horoz, Mehmet; Celik, Yilmaz; Dulger, Ahmet Cumhur; Celik, Hakim; Erel, Ozcan

    2011-06-01

    Myeloperoxidase activity has been investigated after eradication of Helicobacter pylori (H. pylori) in infected patients in previous studies but the results are controversial. The aim of this study was to investigate effect on serum myeloperoxidase activity and oxidative status of eradication treatment in H. pylori-infected patients. Gastric biopsy specimens were obtained from 30 H. pylori infected patients. Serum myeloperoxidase activity was measured by enzyme-linked immunoassay. Oxidative status was determined using total antioxidant capacity (TAC) and total oxidant status (TOS) measurement and calculation of oxidative stress index (OSI). After 2 weeks of the eradication treatment, serum myeloperoxidase activity, TOS and OSI values were significantly lower (all; p<0.001), while TAC was significantly higher (p<0.001). Our results indicate that eradication treatment in H. pylori-infected patients may affect both oxidative stress and myeloperoxidase activity which is an important biomarker in pathogenesis of atherosclerosis. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  12. Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

    PubMed Central

    Falk, R J; Becker, M; Terrell, R; Jennette, J C

    1992-01-01

    We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1379133

  13. Myeloperoxidase-produced Genomic DNA-centered Radicals and Protection by Resveratrol

    EPA Science Inventory

    Myeloperoxidase (MPO) released by activated neutrophils, production of hypochlorous acid (HOCI) and oxidation of the genomic DNA in epithelial cells is thought to initiate and promote carcinogenesis. In this study we applied the 5,5-dimethyl-l-pyrroline N-oxide (DMPO)-based i;nmu...

  14. Effect of Vitamin D Receptor Activation on the AGE/RAGE System and Myeloperoxidase in Chronic Kidney Disease Patients

    PubMed Central

    Pizzini, Patrizia; Cutrupi, Sebastiano; Tripepi, Rocco; Vilasi, Antonio; Tripepi, Giovanni; Mallamaci, Francesca

    2017-01-01

    Vitamin D receptor (VDR) activation has been reported to increase circulating levels of the advanced glycation end products (AGE) and their decoy receptor (RAGE). However, until now, the effect of VDR activation on AGE and RAGE has not been tested in the setting of a randomized, double-blind clinical trial. We have therefore analyzed the effect of VDR activation by paricalcitol on pentosidine, S100A12/ENRAGE, and RAGE and on established biomarkers of oxidative stress like myeloperoxidase in CKD patients in the PENNY trial. At baseline, human S100A12/ENRAGE, RAGE, and myeloperoxidase, but not pentosidine, were intercorrelated, and the association between S100A12/ENRAGE and myeloperoxidase (r = 0.71, P < 0.001) was the strongest among these correlations. Paricalcitol failed to modify biomarkers of the AGE/RAGE system and myeloperoxidase in unadjusted and adjusted analyses by the generalized linear model (GLM). No effect modification by other risk factors was registered. Paricalcitol does not modify biomarkers of the AGE/RAGE system and myeloperoxidase in CKD patients. The apparent increase in RAGE levels by VDR activation reported in previous uncontrolled studies is most likely due to confounding factors rather than to VDR activation per se. This trial is registered with NCT01680198. PMID:29362665

  15. Damage to Candida albicans Hyphae and Pseudohyphae by the Myeloperoxidase System and Oxidative Products of Neutrophil Metabolism In Vitro

    PubMed Central

    Diamond, Richard D.; Clark, Robert A.; Haudenschild, Christian C.

    1980-01-01

    In previous studies, we noted that Candida hyphae and pseudohyphae could be damaged and probably killed by neutrophils, primarily by oxygen-dependent nonphagocytic mechanisms. In extending these studies, amount of damage to hyphae again was measured by inhibition of [14C]cytosine uptake. Neutrophils from only one of four patients with chronic granulomatous disease damaged hyphae at all, and neutrophils from this single patient damaged hyphae far less efficiently than simultaneously tested neutrophils from normal control subjects. Neutrophils from neither of two subjects with hereditary myeloperoxidase deficiency damaged the hyphae. This confirmed the importance of oxidative mechanisms in general and myeloperoxidase-mediated systems in particular in damaging Candida hyphae. Several potentially fungicidal oxidative intermediates are produced by metabolic pathways of normal neutrophils, but their relative toxicity for Candida hyphae was previously unknown. To help determine this, cell-free in vitro systems were used to generate these potentially microbicidal products. Myeloperoxidase with hydrogen peroxide, iodide, and chloride resulted in 91.2% damage to hyphal inocula in 11 experiments. There was less damage when either chloride or iodide was omitted, and no damage when myeloperoxidase was omitted or inactivated by heating. Azide, cyanide, and catalase (but not heated catalase) inhibited the damage. Systems for generation of hydrogen peroxide could replace reagent hydrogen peroxide in the myeloperoxidase system. These included glucose oxidase, in the presence of glucose, and xanthine oxidase, in the presence of either hypoxanthine or acetaldehyde. In the presence of myeloperoxidase and a halide, the toxicity of the xanthine oxidase system was not inhibited by superoxide dismutase and, under some conditions, was marginally increased by this enzyme. This suggested that superoxide radical did not damage hyphae directly but served primarily as an intermediate in the

  16. Leptin protects vital organ functions after sepsis through recovering tissue myeloperoxidase activity: an anti-inflammatory role resonating with indomethacin.

    PubMed

    Lin, Ji; Yan, Guang-Tao; Xue, Hui; Hao, Xiu-Hua; Zhang, Kai; Wang, Lu-Huan

    2007-08-01

    In this research, the role of leptin on sepsis-induced organ dysfunction was evaluated. Making use of a mice sepsis model, changes of alanine transaminase and uric acid in serum, myeloperoxidase activity, leptin levels and histological alterations in heart, lung, liver and kidney were determined. Results showed that sepsis induced significantly higher levels of serum alanine transaminase and uric acid, decreased tissue myeloperoxidase activity and leptin levels, and triggered distinct histological alterations. However, leptin and indomethacin injections reversed those impairments at 6h and/or 12h after injury. These data reveal a protective role of both leptin and indomethacin on vital organ functions after sepsis by recovering tissue myeloperoxidase activity.

  17. Study Identifies New Lymphoma Treatment Target

    Cancer.gov

    NCI researchers have identified new therapeutic targets for diffuse large B-cell lymphoma. Drugs that hit these targets are under clinical development and the researchers hope to begin testing them in clinical trials of patients with DLBCL.

  18. Immune evasion by a staphylococcal inhibitor of myeloperoxidase

    PubMed Central

    de Jong, Nienke W. M.; Ramyar, Kasra X.; Guerra, Fermin E.; Fevre, Cindy; Voyich, Jovanka M.; McCarthy, Alex J.; Garcia, Brandon L.; van Kessel, Kok P. M.; van Strijp, Jos A. G.; Geisbrecht, Brian V.; Haas, Pieter-Jan A.

    2017-01-01

    Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing. PMID:28808028

  19. Immune evasion by a staphylococcal inhibitor of myeloperoxidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    de Jong, Nienke W. M.; Ramyar, Kasra X.; Guerra, Fermin E.

    Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of humanmore » MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.« less

  20. Ethylene formation by polymorphonuclear leukocytes. Role of myeloperoxidase

    PubMed Central

    1978-01-01

    Ethylene formation from the thioethers, beta-methylthiopropionaldehyde (methional) and 2-keto-4-thiomethylbutyric acid by phagocytosing polymorphonuclear leukocytes (PMNs) was found to be largely dependent on myeloperoxidase (MPO). Conversion was less than 10% of normal when MPO-deficient PMNs were employed; formation by normal PMNs was inhibited by the peroxidase inhibitors, azide, and cyanide, and a model system consisting of MPO, H2O2, chloride (or bromide) and EDTA was found which shared many of the properties of the predominant PMN system. MPO-independent mechanisms of ethylene formation were also identified. Ethylene formation from methional by phagocytosing eosinophils and by H2O2 in the presence or absence of catalase was stimulated by azide. The presence of MPO-independent, azide-stimulable systems in the PMN preparations was suggested by the azide stimulation of ethylene formation from methional when MPO-deficient leukocytes were employed. Ethylene formation by dye-sensitized photooxidation was also demonstrated and evidence obtained for the involvement of singlet oxygen (1O2). These findings are discussed in relation to the participation of H2O2, hydroxyl radicals, the superoxide anion and 1O2 in the formation of ethylene by PMNs and by the MPO model system. PMID:212502

  1. Cot/tpl2 (MAP3K8) mediates myeloperoxidase activity and hypernociception following peripheral inflammation.

    PubMed

    Soria-Castro, Irene; Krzyzanowska, Agnieszka; Pelaéz, Marta López; Regadera, Javier; Ferrer, Gema; Montoliu, Lluis; Rodríguez-Ramos, Rosario; Fernández, Margarita; Alemany, Susana

    2010-10-29

    Cot/tpl2 (also known as MAP3K8) has emerged as a new and potentially interesting therapeutic anti-inflammatory target. Here, we report the first study of Cot/tpl2 involvement in acute peripheral inflammation in vivo. Six hours after an intraplantar injection of zymosan, Cot/tpl2(-/-) mice showed a 47% reduction in myeloperoxidase activity, concomitant with a 46% lower neutrophil recruitment and a 40% decreased luminol-mediated bioluminescence imaging in vivo. Accordingly, Cot/tpl2 deficiency provoked a 25-30% reduction in luminol-mediated bioluminescence and neutrophil recruitment together with a 65% lower macrophage recruitment 4 h following zymosan-induced peritonitis. Significantly impaired levels of G-CSF and GM-CSF and of other cytokines such as TNFα, IL-1β, and IL-6, as well as some chemokines such as MCP-1, MIP-1β, and keratinocyte-derived chemokine, were detected during the acute zymosan-induced intraplantar inflammatory response in Cot/tpl2(-/-) mice. Moreover, Cot/tpl2 deficiency dramatically decreased the production of the hypernociceptive ligand NGF at the inflammatory site during the course of inflammation. Most importantly, Cot/tpl2 deficiency significantly reduced zymosan-induced inflammatory hypernociception in mice, with a most pronounced effect of a 50% decrease compared with wild type (WT) at 24 h following intraplantar injection of zymosan. At this time, Cot/tpl2(-/-) mice showed significantly reduced NGF, TNFα, and prostaglandin E(2) levels compared with WT littermates. In conclusion, our study demonstrates an important role of Cot/tpl2 in the NGF, G-CSF, and GM-CSF production and myeloperoxidase activity in the acute inflammatory response process and its implication in inflammatory hypernociception.

  2. Biosynthesis of human myeloperoxidase.

    PubMed

    Nauseef, William M

    2018-03-15

    Members of Chordata peroxidase subfamily [1] expressed in mammals, including myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO), express conserved motifs around the heme prosthetic group essential for their activity, a calcium-binding site, and at least two covalent bonds linking the heme group to the protein backbone. Although most studies of the biosynthesis of these peroxidases have focused on MPO, many of the features described occur during biosynthesis of other members of the protein subfamily. Whereas MPO biosynthesis includes events typical for proteins generated in the secretory pathway, the importance and consequences of heme insertion are events uniquely associated with peroxidases. This Review summarizes decades of work elucidating specific steps in the biosynthetic pathway of human MPO. Discussion includes cotranslational glycosylation and subsequent modifications of the N-linked carbohydrate sidechains, contributions by molecular chaperones in the endoplasmic reticulum, cleavage of the propeptide from proMPO, and proteolytic processing of protomers and dimerization to yield mature MPO. Parallels between the biosynthesis of MPO and TPO as well as the impact of inherited mutations in the MPO gene on normal biosynthesis will be summarized. Lastly, specific gaps in our knowledge revealed by this review of our current understanding will be highlighted. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. [Cytochemical parameters of myeloperoxidase activity and catecholamine level in blood of postpartum women living in areas near the Semipalatinsk nuclear test site].

    PubMed

    Kokabaeva, A E; Bazeliuk, L T

    2002-01-01

    The activity of neitrophil myeloperoxidase and content of blood etyrhrocyte cathecholamines in the blood of women in early postpartum period in dependence on distance of their living area from Semipalatinsk nuclear testing were studied. It was found that women who live closer to Semipalatinsk were characterised by significantly lower neitrophil myeloperoxidase activity and content of cathecholamines in erythrocytes than in control.

  4. Use of polyclonal anti-myeloperoxidase antibody in myeloid lineage determination.

    PubMed

    Karnik, M P; Nair, C N; Zingde, S M; Gothoskar, B P; Zachariah, L; Barbhaya, S; Advani, S H

    1994-12-01

    This study reports the production of a rabbit polyclonal antibody to myeloperoxidase (MPO) and its use in ascertaining the myeloid lineage of blasts in leukaemia. Comparison of the immunocytochemical stain using the anti-MPO antibody with the routine cytochemical methodology showed that the former was more sensitive. In all subtypes of acute myeloid leukaemia (AML; 72 patients, M1-M6) greater number of MPO positive blast cells were observed by immunocytochemistry, the highest being in the promyelocytic leukaemia. It was also extremely specific for cells of the myeloid lineage as it did not react with blasts from acute lymphoblastic (50 patients) and megakaryoblastic leukaemias (1 patient). In addition, it proved most useful for the lineage determination of blasts from patients with undifferentiated acute leukaemias (AUL) and those with chronic myeloid leukaemia in blast crisis (CML-BC). Out of 8 patients of AULs, 6 were classified as acute myeloblastic leukaemia due to their reactivity to the anti-MPO antibody. Similarly, out of 12 patients of chronic myeloid leukaemia in blast crisis, blasts from 8 showed reactivity to this antibody and thus could be identified as belonging to the myeloid lineage and/or of the mixed blast crisis type.

  5. The molecular mechanism for interaction of ceruloplasmin and myeloperoxidase

    NASA Astrophysics Data System (ADS)

    Bakhautdin, Bakytzhan; Bakhautdin, Esen Göksöy

    2016-04-01

    Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.

  6. Assessment of myeloperoxidase activity in renal tissue after ischemia/reperfusion.

    PubMed

    Laight, D W; Lad, N; Woodward, B; Waterfall, J F

    1994-11-01

    We have shown that a photometric assay of myeloperoxidase derived from rat blood polymorphonucleocytes employing 3,3',5,5'-tetramethylbenzidine as substrate is more sensitive than an established assay employing o-dianisidine. We went on to demonstrate that rat renal tissue is capable of inhibiting peroxidase activity. This activity approached 100% when the rat renal supernate was incubated at 60 degree C for 2 h and the assay was conducted in the presence of a 10-fold higher concentration of hydrogen peroxide (H2O2). Rat kidneys undergoing 45 min ischaemia and 1,3 and 6 h reperfusion in vivo, exhibited significant increases in myeloperoxidase activity, indicating tissue polymorphonucleocyte accumulation. Monoclonal antibodies against rat intercellular adhesion molecule 1 (ICAM-1) and CD18 of beta 2-integrins administered both 5 min before a period of 45 min renal ischaemia (20 micrograms/kg i.v.) and at the commencement of 1 h reperfusion (20 micrograms/kg i.v.) reduced renal tissue polymorphonucleocyte accumulation. However, similar treatment with the parent murine antibody immunoglobulin G1 (IgG1) and an unrelated murine antibody, IgG2a, also significantly reduced renal tissue polymorphonucleocyte accumulation. In conclusion, we demonstrate that the rat renal suppression of peroxidase activity can be overcome by a combination of heat inactivation and the provision of excess assay H2O2. In addition, the available evidence suggests that murine monoclonal antibodies against rat adhesion molecules may exert non-specific actions in our model of renal ischaemia/reperfusion in vivo.

  7. Myeloperoxidase-hydrogen peroxide-chloride antimicrobial system: effect of exogenous amines on antibacterial action against Escherichia coli.

    PubMed

    Thomas, E L

    1979-07-01

    Exogenous ammonium ions (NH(4) (+)) and amine compounds had a profound influence on the antibacterial activity of the myeloperoxidase-hydrogen peroxide-chloride system against Escherichia coli. The rate of killing increased in the presence of NH(4) (+) and certain guanidino compounds and decreased in the presence of alpha-amino acids, polylysine, taurine, or tris (hydroxymethyl) aminomethane. Myeloperoxidase catalyzed the oxidation of chloride to hypochlorous acid, which reacted either with bacterial amine or amide components or both or with the exogenous compounds to yield chloramine or chloramide derivatives or both. These nitrogen-chlorine derivatives could oxidize bacterial components. Killing was correlated with oxidation of bacterial components. The rate of oxidation of bacterial sulfhydryls increased in the presence of the compounds that increased the rate of killing and decreased in the presence of the other compounds. The reaction of HOCl with NH(4) (+) yielded monochloramine (NH(2)Cl), which could be extracted into organic solvents. The N-Cl derivatives of bacterial components or of polylysine, taurine, or tris(hydroxymethyl)aminomethane could not be extracted. The effect of NH(4) (+) on killing is attributed to the ability of NH(2)Cl to penetrate the hydrophobic cell membrane and thus to oxidize intracellular components. Polylysine, taurine, and tris(hydroxymethyl)aminomethane formed high-molecular-weight, charged, or polar N-Cl derivatives that would be unable to penetrate the cell membrane. These results suggest an important role for leukocyte amine components in myeloperoxidase-catalyzed antimicrobial activity in vivo.

  8. Myeloperoxidase as an Active Disease Biomarker: Recent Biochemical and Pathological Perspectives.

    PubMed

    Khan, Amjad A; Alsahli, Mohammed A; Rahmani, Arshad H

    2018-04-18

    Myeloperoxidase (MPO) belongs to the family of heme-containing peroxidases, produced mostly from polymorphonuclear neutrophils. The active enzyme (150 kDa) is the product of the MPO gene located on long arm of chromosome 17. The primary gene product undergoes several modifications, such as the removal of introns and signal peptides, and leads to the formation of enzymatically inactive glycosylated apoproMPO which complexes with chaperons, producing inactive proMPO by the insertion of a heme moiety. The active enzyme is a homodimer of heavy and light chain protomers. This enzyme is released into the extracellular fluid after oxidative stress and different inflammatory responses. Myeloperoxidase is the only type of peroxidase that uses H₂O₂ to oxidize several halides and pseudohalides to form different hypohalous acids. So, the antibacterial activities of MPO involve the production of reactive oxygen and reactive nitrogen species. Controlled MPO release at the site of infection is of prime importance for its efficient activities. Any uncontrolled degranulation exaggerates the inflammation and can also lead to tissue damage even in absence of inflammation. Several types of tissue injuries and the pathogenesis of several other major chronic diseases such as rheumatoid arthritis, cardiovascular diseases, liver diseases, diabetes, and cancer have been reported to be linked with MPO-derived oxidants. Thus, the enhanced level of MPO activity is one of the best diagnostic tools of inflammatory and oxidative stress biomarkers among these commonly-occurring diseases.

  9. TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

    PubMed

    Xu, Yan; Chen, Yan; Li, Daliang; Liu, Qing; Xuan, Zhenyu; Li, Wen-Hong

    2017-02-01

    MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

  10. Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue.

    PubMed

    Turunen, Sanna; Huhtakangas, Johanna; Nousiainen, Tomi; Valkealahti, Maarit; Melkko, Jukka; Risteli, Juha; Lehenkari, Petri

    2016-10-20

    Seropositive rheumatoid arthritis (RA) is characterized by autoantibodies binding to citrullinated and homocitrullinated proteins. We wanted to study the expression patterns of these disease-associated protein forms and if the rheumatoid nodule and synovial tissue itself contain biologically active levels of citrullinating peptidyl arginine deiminases 2, 3 and 4 and homocitrullination-facilitating neutrophil enzyme myeloperoxidase. Total of 195 synovial samples from metatarsal joints from five ACPA/RF-positive RA patients (n = 77), synovial samples from knees of eight seropositive RA (n = 60), seven seronegative RA (n = 33) and five osteoarthritis (n = 25) patients were analyzed for citrulline and homocitrulline contents using HPLC. The location of citrulline- and homocitrulline-containing proteins, PAD 2, 3, 4 and myeloperoxidase were shown by immunostaining. Myeloperoxidase and citrulline- or homocitrulline-containing proteins were stained on Western blot. Overall, necrosis was frequent in metatarsals of seropositive RA and absent in seronegative RA and osteoarthritis patients. In histological analysis, there was a significant local patterning and variation in the citrulline and homocitrulline content and it was highest in metatarsal synovial tissues of seropositive RA patients. We found peptidyl arginine deiminase 2, 3 and 4 in the lining and sublining layers of intact synovial tissue. Myeloperoxidase was found locally around necrotic areas. The tissues with necrosis contained the highest levels of citrulline and homocitrulline. Rheumatoid nodules and synovia contain significant amount of PAD2, 3 and 4 and myeloperoxidase enzymes. These enzymes could explain the levels of citrulline and homocitrulline in seropositive RA synovial and rheumatoid nodule tissues especially around necrotic tissue.

  11. Identifying transcription factor functions and targets by phenotypic activation

    PubMed Central

    Chua, Gordon; Morris, Quaid D.; Sopko, Richelle; Robinson, Mark D.; Ryan, Owen; Chan, Esther T.; Frey, Brendan J.; Andrews, Brenda J.; Boone, Charles; Hughes, Timothy R.

    2006-01-01

    Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology. PMID:16880382

  12. 2-Thioxanthines Are Mechanism-based Inactivators of Myeloperoxidase That Block Oxidative Stress during Inflammation*

    PubMed Central

    Tidén, Anna-Karin; Sjögren, Tove; Svensson, Mats; Bernlind, Alexandra; Senthilmohan, Revathy; Auchère, Francoise; Norman, Henrietta; Markgren, Per-Olof; Gustavsson, Susanne; Schmidt, Staffan; Lundquist, Stefan; Forbes, Louisa V.; Magon, Nicholas J.; Paton, Louise N.; Jameson, Guy N. L.; Eriksson, Håkan; Kettle, Anthony J.

    2011-01-01

    Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation. PMID:21880720

  13. A biochemical approach to identifying microRNA targets

    PubMed Central

    Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

    2007-01-01

    Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. PMID:18042700

  14. Selecting a Targeting Method to Identify BPL Households in India

    ERIC Educational Resources Information Center

    Alkire, Sabina; Seth, Suman

    2013-01-01

    This paper proposes how to select a methodology to target multidimensionally poor households, and how to update that targeting exercise periodically. We present this methodology in the context of discussions regarding the selection of a targeting methodology in India. In 1992, 1997, and 2002 the Indian government identified households that are…

  15. [Morphological characteristics of inflammation in HIV-associated pulmonary tuberculosis with regard to the expression of myeloperoxidase].

    PubMed

    Bykhalov, L S; Smirnov, A V

    2015-01-01

    to characterize the morphological features of inflammation and the degree of myeloperoxidase expression in the lung of patients who died from HIV-associated tuberculosis (TB). Autopsy lung tissue specimens from 229 patients with HIV-associated TB were examined. A comparison group consisted of dead TB mono-infected patients (n = 30). Dead HIV/TB co-infected patients were divided into subgroups: 1) 50 patients with Stage 4A-4B and a CD4(+)-lymphocyte count of more than 200 cells/µl; 2) 54 with Stage 4B-4C and a CD4(+)-lymphocyte count of 100 to 200 cells/µl; 3) 125 with Stage 4C-5 and a CD4(+)-lymphocyte count of less than 100 cells/µl. Histological and immunohistochemical examinations of lung slices were performed using antibodies to myeloperoxidase (MPO). The predominant types of an inflammatory response were revealed according to the level of CD4+ lymphocytes. The lungs in Subgroup 1 showed a predominant typical granulomatous response (82%). Subgroup 2 exhibited an exudative-productive inflammatory response (57.4%) while Subgroup 3 displayed an alterative necrotic type (92.8%). Subgroup 3 showed the most marked reduction in lymphocyte numbers in the areas of inflammation, which was accompanied by a significant increase in the relative density and other morphometric parameters of MPO-positive macrophages and granulocytes in the inflammatory infiltration zones and alveolar walls. The identified changes in the lungs suggest that there is a decline in the magnitude of a delayed type hypersensitivity reaction, a progression of alterative necrotic processes as CD4(+) lymphocytes decrease in the systemic blood flow, and an increase in the proportion of functionally immature macrophages in the areas of inflammation, which reflects a substantially impaired local immune response to the persistence of M. tuberculosis in HIV infection.

  16. UniDrug-target: a computational tool to identify unique drug targets in pathogenic bacteria.

    PubMed

    Chanumolu, Sree Krishna; Rout, Chittaranjan; Chauhan, Rajinder S

    2012-01-01

    Targeting conserved proteins of bacteria through antibacterial medications has resulted in both the development of resistant strains and changes to human health by destroying beneficial microbes which eventually become breeding grounds for the evolution of resistances. Despite the availability of more than 800 genomes sequences, 430 pathways, 4743 enzymes, 9257 metabolic reactions and protein (three-dimensional) 3D structures in bacteria, no pathogen-specific computational drug target identification tool has been developed. A web server, UniDrug-Target, which combines bacterial biological information and computational methods to stringently identify pathogen-specific proteins as drug targets, has been designed. Besides predicting pathogen-specific proteins essentiality, chokepoint property, etc., three new algorithms were developed and implemented by using protein sequences, domains, structures, and metabolic reactions for construction of partial metabolic networks (PMNs), determination of conservation in critical residues, and variation analysis of residues forming similar cavities in proteins sequences. First, PMNs are constructed to determine the extent of disturbances in metabolite production by targeting a protein as drug target. Conservation of pathogen-specific protein's critical residues involved in cavity formation and biological function determined at domain-level with low-matching sequences. Last, variation analysis of residues forming similar cavities in proteins sequences from pathogenic versus non-pathogenic bacteria and humans is performed. The server is capable of predicting drug targets for any sequenced pathogenic bacteria having fasta sequences and annotated information. The utility of UniDrug-Target server was demonstrated for Mycobacterium tuberculosis (H37Rv). The UniDrug-Target identified 265 mycobacteria pathogen-specific proteins, including 17 essential proteins which can be potential drug targets. UniDrug-Target is expected to accelerate

  17. Identifying Molecular Targets for PTSD Treatment Using Single Prolonged Stress

    DTIC Science & Technology

    2015-10-01

    1 AWARD NUMBER: W81XWH-13-1-0377 TITLE: Identifying Molecular Targets For PTSD Treatment Using Single Prolonged Stress PRINCIPAL...TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-13-1-0377 Identifying Molecular Targets For PTSD Treatment Using Single Prolonged Stress 5b. GRANT...brain GR and β-AR expression alters glutamatergic and GABAergic function in neural circuits that mediate SPS-induced deficits in extinction retention

  18. In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

    PubMed

    Hülsdünker, Jan; Zeiser, Robert

    2016-01-01

    Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.

  19. Myeloperoxidase-induced Genomic DNA-centered Radicals*

    PubMed Central

    Gomez-Mejiba, Sandra E.; Zhai, Zili; Gimenez, Maria S.; Ashby, Michael T.; Chilakapati, Jaya; Kitchin, Kirk; Mason, Ronald P.; Ramirez, Dario C.

    2010-01-01

    Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis. PMID:20406811

  20. Relationship between the lipophilicity of gallic acid n-alquil esters' derivatives and both myeloperoxidase activity and HOCl scavenging.

    PubMed

    Rosso, Rober; Vieira, Tiago O; Leal, Paulo C; Nunes, Ricardo J; Yunes, Rosendo A; Creczynski-Pasa, Tânia B

    2006-09-15

    The gallic acid and several n-alkyl gallates, with the same number of hydroxyl substituents, varying only in the side carbonic chain length, with respective lipophilicity defined through the C log P, were studied. It evidenced the structure-activity relationship of the myeloperoxidase activity inhibition and the hypochlorous acid scavenger property, as well as its low toxicity in rat hepatic tissue. The gallates with C log P below 3.0 (compounds 2-7) were more active against the enzyme activity, what means that the addition of 1-6 carbons (C log P between 0.92 and 2.92) at the side chain increased approximately 50% the gallic acid effect. However, a relationship between the HOCl scavenging capability and the lipophilicity was not observed. With these results it is possible to suggest that the gallates protect the HOCl targets through two mechanisms: inhibiting its production by the enzyme and scavenging the reactive specie.

  1. Evidence that a formyl-substituted iron porphyrin is the prosthetic group of myeloperoxidase: magnetic circular dichroism similarity of the peroxidase to Spirographis heme-reconstituted myoglobin.

    PubMed Central

    Sono, M; Bracete, A M; Huff, A M; Ikeda-Saito, M; Dawson, J H

    1991-01-01

    To probe the identity of the active site heme-type prosthetic group of myeloperoxidase, whose structure has not been established unambiguously [proposed structures are (i) a chlorin (dihydroporphyrin) or (ii) a formyl-substituted porphyrin such as present in heme a], Spirographis heme (2-formyl-4-vinyldeuteroheme IX) has been incorporated into apo-myoglobin as a possible iron porphyrin model. Comparison of parallel derivatives of these two green proteins with magnetic circular dichroism spectroscopy reveals considerable similarities between several derivatives of these proteins, including the pyridine hemochromogen, the native ferric, ferrous-oxy, and ferrous-CO forms. In contrast, the magnetic circular dichroism spectra of available iron chlorin (octaethylchlorin) model complexes in analogous ligation and oxidation states do not show any significant spectral similarities to myeloperoxidase. This finding provides important evidence in favor of a formyl-substituted porphyrin as the structure of the prosthetic group macrocycle of myeloperoxidase. PMID:1662385

  2. Anti-Myeloperoxidase Antibodies Associate with Future Proliferative Lupus Nephritis

    PubMed Central

    Lee, J. J.; Poirier, M.; Little, D. J.; Prince, L. K.; Baker, T. P.; Edison, J. D.; Abbott, K. C.

    2017-01-01

    Background The subclinical pathophysiology of proliferative lupus nephritis (PLN) has not been fully elucidated. Myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA) is associated with PLN, but prediagnostic levels have not been reported. Methods We performed a retrospective case-control Department of Defense Serum Repository (DoDSR) study comparing MPO-ANCA levels in longitudinal prediagnostic serum samples for 23 biopsy confirmed proliferative lupus nephritis (PLN) patients to DoDSR identified age, sex, race, and age of serum matched healthy and SLE without LN disease controls. We also compared the temporal relationship of MPO-ANCA to anti-double stranded DNA antibodies (dsDNAab). Results A greater proportion of PLN patients had prediagnostic MPO-ANCA levels above ≥3 U/mL and ≥6 U/mL compared to SLE without LN (91% versus 43%, p < 0.001; 57% versus 5%, p < 0.001, resp.). In subgroup analysis, the MPO-ANCA threshold of ≥3 U/mL was significant at <1 year (88% versus 39%, p = 0.007) and 1–4 years (87% versus 38%, p = 0.009) prior to diagnosis. Statistically significant subclinical MPO-ANCA levels (≥3 U/mL) occurred prior to statistically significant dsDNAab ≥ 3 IU/ml (89% versus 11%, p = 0.003). Conclusions Subclinical MPO-ANCA levels could distinguish future PLN from SLE without LN. MPO-ANCA manifests prior to clinical disease and subclinical dsDNAab to suggest that it may contribute directly to PLN pathogenicity. PMID:29435367

  3. A side-effect free method for identifying cancer drug targets.

    PubMed

    Ashraf, Md Izhar; Ong, Seng-Kai; Mujawar, Shama; Pawar, Shrikant; More, Pallavi; Paul, Somnath; Lahiri, Chandrajit

    2018-04-27

    Identifying effective drug targets, with little or no side effects, remains an ever challenging task. A potential pitfall of failing to uncover the correct drug targets, due to side effect of pleiotropic genes, might lead the potential drugs to be illicit and withdrawn. Simplifying disease complexity, for the investigation of the mechanistic aspects and identification of effective drug targets, have been done through several approaches of protein interactome analysis. Of these, centrality measures have always gained importance in identifying candidate drug targets. Here, we put forward an integrated method of analysing a complex network of cancer and depict the importance of k-core, functional connectivity and centrality (KFC) for identifying effective drug targets. Essentially, we have extracted the proteins involved in the pathways leading to cancer from the pathway databases which enlist real experimental datasets. The interactions between these proteins were mapped to build an interactome. Integrative analyses of the interactome enabled us to unearth plausible reasons for drugs being rendered withdrawn, thereby giving future scope to pharmaceutical industries to potentially avoid them (e.g. ESR1, HDAC2, F2, PLG, PPARA, RXRA, etc). Based upon our KFC criteria, we have shortlisted ten proteins (GRB2, FYN, PIK3R1, CBL, JAK2, LCK, LYN, SYK, JAK1 and SOCS3) as effective candidates for drug development.

  4. Genetic variation in the myeloperoxidase gene and cognitive impairment in Multiple Sclerosis

    PubMed Central

    Manna, I; Valentino, P; La Russa, A; Condino, F; Nisticò, R; Liguori, M; Clodomiro, A; Andreoli, V; Pirritano, D; Cittadella, R; Quattrone, A

    2006-01-01

    There is evidence that multiple sclerosis (MS) may associated with cognitive impairment in 25 to 40% of cases. The gene encoding myeloperoxidase (MPO) is involved in molecular pathways leading to β-amyloid deposition. We investigated a functional biallelic (G/A) polymorphism in the promoter region (-463) of the MPO gene in 465 patients affected by MS, divided into 204 cognitively normal and 261 impaired. We did not find significant differences in allele or genotype distributions between impaired and preserved MS patients. Our findings suggest that MPO polymorphism is not a risk factor for cognitive impairment in MS. PMID:16504169

  5. Geothermal Target Areas in Colorado as Identified by Remote Sensing Techniques

    DOE Data Explorer

    Khalid Hussein

    2012-02-01

    This layer contains the areas identified as targets of potential geothermal activity. The Criteria used to identify the target areas include: hot/warm surface exposures modeled from ASTER/Landsat satellite imagery and geological characteristics, alteration mineral commonly associated with hot springs (clays, Si, and FeOx) modeled from ASTER and Landsat data, Colorado Geological Survey (CGS) known thermal hot springs/wells and heat-flow data points, Colorado deep-seated fault zones, weakened basement identified from isostatic gravity data, and Colorado sedimentary and topographic characteristics.

  6. Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells.

    PubMed

    Mishra, Om P; Popov, Anatoliy V; Pietrofesa, Ralph A; Nakamaru-Ogiso, Eiko; Andrake, Mark; Christofidou-Solomidou, Melpo

    2018-06-01

    Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl - . EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Neutrophils differentially attenuate immune response to Aspergillus infection through complement receptor 3 and induction of myeloperoxidase.

    PubMed

    Goh, Jessamine G; Ravikumar, Sharada; Win, Mar Soe; Cao, Qiong; Tan, Ai Ling; Lim, Joan H J; Leong, Winnie; Herbrecht, Raoul; Troke, Peter F; Kullberg, Bart Jan; Netea, Mihai G; Chng, Wee Joo; Dan, Yock Young; Chai, Louis Y A

    2018-03-01

    Invasive aspergillosis (IA) remains a major cause of morbidity in immunocompromised hosts. This is due to the inability of the host immunity to respond appropriately to Aspergillus. An established risk factor for IA is neutropenia that is encountered by patients undergoing chemotherapy. Herein, we investigate the role of neutrophils in modulating host response to Aspergillus. We found that neutrophils had the propensity to suppress proinflammatory cytokine production but through different mechanisms for specific cytokines. Cellular contact was requisite for the modulation of interleukin-1 beta production by Aspergillus with the involvement of complement receptor 3. On the other hand, inhibition of tumour necrosis factor-alpha production (TNF-α) was cell contact-independent and mediated by secreted myeloperoxidase. Specifically, the inhibition of TNF-α by myeloperoxidase was through the TLR4 pathway and involved interference with the mRNA transcription of TNF receptor-associated factor 6/interferon regulatory factor 5. Our study illustrates the extended immune modulatory role of neutrophils beyond its primary phagocytic function. The absence of neutrophils and loss of its inhibitory effect on cytokine production explains the hypercytokinemia seen in neutropenic patients when infected with Aspergillus. © 2017 John Wiley & Sons Ltd.

  8. Effect of g.9476869G>A myeloperoxidase (MPO) gene polymorphism on the antioxidant activity of milk from Polish Holstein-Friesian cows of the Black-and-White variety (HO).

    PubMed

    Pokorska, Joanna; Poskart, Karolina; Kułaj, Dominika; Ochrem, Andrzej; Dusza, Magdalena; Gil, Zygmunt; Świętek, Ewa; Makulska, Joanna

    2017-05-01

    Myeloperoxidase (MPO) is an important enzyme, which is one of the components of the antibacterial system in neutrophils and monocytes. MPO participates in the inflammatory response in multiple locations in the body, including the mammary glands. As a result of the activity of MPO, many oxidising compounds as well as reactive oxygen species are generated. It seems that myeloperoxidase may be a marker linking inflammation processes and oxidative stress. So far, there are no literature data on the association between the MPO gene polymorphism and the antioxidant properties of milk. The aim of the study was to analyse the effect of g.9476869G > A polymorphism of myeloperoxidase (MPO) gene and age of cows on the antioxidant activity of milk and other milk traits in Polish Holstein-Friesian cows. Polymorphism of MPO gene was identified by the PCR-RFLP method using the HphI endonuclease. The total antioxidant capacity of milk samples was measured by the Trolox Equivalent Antioxidant Capacity (TEAC) method. It was found that the GG genotype was the most frequent (0·606). The genotype at the tested MPO locus and the age of the animals affected the antioxidant activity of milk. Milk from cows with the GA genotype was characterised by a significantly higher antioxidant activity than milk from cows with the GG genotype (P < 0·0001). The analysis of interaction showed that cows with the GA genotype and older than 6·5 years produced milk with a significantly higher antioxidant activity compared with younger cows with the same genotype (P < 0·0001), as well as cows with the GG genotype of all ages (P < 0·0001).

  9. A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

    DTIC Science & Technology

    2004-05-01

    AD Award Number: DAMD17-03-1-0232 TITLE: A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast PRINCIPAL INVESTIGATOR...Approach to Identify Novel Breast DAMD17-03-1-0232 Cancer Gene Targets in Yeast 6. A UTHOR(S) Craig Bennett, Ph.D. 7. PERFORMING ORGANIZA TION NAME(S...Unlimited 13. ABSTRACT (Maximum 200 Words) We are using the yeast Saccharomyces cerevisiae to identify new cancer gene targets that interact with the

  10. Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions.

    PubMed Central

    Hazen, S L; Gaut, J P; Crowley, J R; Hsu, F F; Heinecke, J W

    2000-01-01

    Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein. PMID:11104675

  11. Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells.

    PubMed Central

    Savage, C. O.; Pottinger, B. E.; Gaskin, G.; Pusey, C. D.; Pearson, J. D.

    1992-01-01

    The ability of vasculitis-associated anti-neutrophil cytoplasm antibodies (ANCA) to activate neutrophils and mediate release of radiolabel from 111Indium-labeled cultured human umbilical vein endothelial cells (HUVEC) was determined as a measure of the potential cytotoxicity of ANCA-activated neutrophils against vascular endothelium. Priming of neutrophils with low doses of phorbol 12-myristate 13-acetate (PMA) (1 ng/ml) and ionomycin (0.1 mumol/1) was required, together with pretreatment of endothelial cells with BCNU (1,3-bis-[2-chloroethyl]-1-nitrosourea; 0.26 mmol/l). Under these conditions and using a 4-hour serum-free assay system, mouse monoclonal antibodies (MAb) to the target autoantigens proteinase-3 (Pr-3) and myeloperoxidase (MPO) mediated enhanced release of 111Indium from HUVEC compared with control MAb. Human IgG Fab2 C-ANCA (recognizing Pr-3) and P-ANCA (recognizing MPO) did likewise. Preactivation of HUVEC with TNF (50 U/ml, 4 hr) enhanced the release of 111Indium from HUVEC generated by neutrophils activated with anti-Pr-3 and anti-MPO MAb. These data support the suggestion that activation of neutrophils by ANCA within the vascular lumen may contribute to endothelial cell injury. PMID:1323218

  12. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    PubMed

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  13. Functional kinomics identifies candidate therapeutic targets in head and neck cancer

    PubMed Central

    Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M.; Gurley, Kay E.; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G.; Margolin, Adam A.; Grandori, Carla; Kemp, Christopher J.; Méndez, Eduardo

    2014-01-01

    Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. PMID:25125259

  14. Functional kinomics identifies candidate therapeutic targets in head and neck cancer.

    PubMed

    Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M; Gurley, Kay E; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G; Margolin, Adam A; Grandori, Carla; Kemp, Christopher J; Méndez, Eduardo

    2014-08-15

    To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. ©2014 American Association for Cancer Research.

  15. Urate as a Physiological Substrate for Myeloperoxidase

    PubMed Central

    Meotti, Flavia C.; Jameson, Guy N. L.; Turner, Rufus; Harwood, D. Tim; Stockwell, Samantha; Rees, Martin D.; Thomas, Shane R.; Kettle, Anthony J.

    2011-01-01

    Urate and myeloperoxidase (MPO) are associated with adverse outcomes in cardiovascular disease. In this study, we assessed whether urate is a likely physiological substrate for MPO and if the products of their interaction have the potential to exacerbate inflammation. Urate was readily oxidized by MPO and hydrogen peroxide to 5-hydroxyisourate, which decayed to predominantly allantoin. The redox intermediates of MPO were reduced by urate with rate constants of 4.6 × 105 m−1 s−1 for compound I and 1.7 × 104 m−1 s−1 for compound II. Urate competed with chloride for oxidation by MPO and at hyperuricemic levels is expected to be a substantive substrate for the enzyme. Oxidation of urate promoted super-stoichiometric consumption of glutathione, which indicates that it is converted to a free radical intermediate. In combination with superoxide and hydrogen peroxide, MPO oxidized urate to a reactive hydroperoxide. This would form by addition of superoxide to the urate radical. Urate also enhanced MPO-dependent consumption of nitric oxide. In human plasma, stimulated neutrophils produced allantoin in a reaction dependent on the NADPH oxidase, MPO and superoxide. We propose that urate is a physiological substrate for MPO that is oxidized to the urate radical. The reactions of this radical with superoxide and nitric oxide provide a plausible link between urate and MPO in cardiovascular disease. PMID:21266577

  16. Association of Osteopontin, Neopterin, and Myeloperoxidase With Stroke Risk in Patients With Prior Stroke or Transient Ischemic Attacks: Results of an Analysis of 13 Biomarkers From the Stroke Prevention by Aggressive Reduction in Cholesterol Levels Trial.

    PubMed

    Ganz, Peter; Amarenco, Pierre; Goldstein, Larry B; Sillesen, Henrik; Bao, Weihang; Preston, Gregory M; Welch, K Michael A

    2017-12-01

    Established risk factors do not fully identify patients at risk for recurrent stroke. The SPARCL trial (Stroke Prevention by Aggressive Reduction in Cholesterol Levels) evaluated the effect of atorvastatin on stroke risk in patients with a recent stroke or transient ischemic attack and no known coronary heart disease. This analysis explored the relationships between 13 plasma biomarkers assessed at trial enrollment and the occurrence of outcome strokes. We conducted a case-cohort study of 2176 participants; 562 had outcome strokes and 1614 were selected randomly from those without outcome strokes. Time to stroke was evaluated by Cox proportional hazards models. There was no association between time to stroke and lipoprotein-associated phospholipase A 2 , monocyte chemoattractant protein-1, resistin, matrix metalloproteinase-9, N-terminal fragment of pro-B-type natriuretic peptide, soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1, or soluble CD40 ligand. In adjusted analyses, osteopontin (hazard ratio per SD change, 1.362; P <0.0001), neopterin (hazard ratio, 1.137; P =0.0107), myeloperoxidase (hazard ratio, 1.177; P =0.0022), and adiponectin (hazard ratio, 1.207; P =0.0013) were independently associated with outcome strokes. After adjustment for the Stroke Prognostic Instrument-II and treatment, osteopontin, neopterin, and myeloperoxidase remained independently associated with outcome strokes. The addition of these 3 biomarkers to Stroke Prognostic Instrument-II increased the area under the receiver operating characteristic curve by 0.023 ( P =0.015) and yielded a continuous net reclassification improvement (29.1%; P <0.0001) and an integrated discrimination improvement (42.3%; P <0.0001). Osteopontin, neopterin, and myeloperoxidase were independently associated with the risk of recurrent stroke and improved risk classification when added to a clinical risk algorithm. URL: http://www.clinicaltrials.gov. Unique Identifier: NCT

  17. Using Click Chemistry to Identify Potential Drug Targets in Plasmodium

    DTIC Science & Technology

    2015-04-01

    step of the Plasmodium mammalian cycle . Inhibiting this step can block malaria at an early step. However, few anti-malarials target liver infection...points in the life cycle of malaria parasites. PLoS Biol 12: e1001806. 2. Falae A, Combe A, Amaladoss A, Carvalho T, Menard R, et al. (2010) Role of...AWARD NUMBER: W81XWH-13-1-0429 TITLE: Using "Click Chemistry" to Identify Potential Drug Targets in Plasmodium PRINCIPAL INVESTIGATOR: Dr. Purnima

  18. Identifying protein kinase target preferences using mass spectrometry

    PubMed Central

    Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

    2012-01-01

    A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

  19. Integrative biology approach identifies cytokine targeting strategies for psoriasis.

    PubMed

    Perera, Gayathri K; Ainali, Chrysanthi; Semenova, Ekaterina; Hundhausen, Christian; Barinaga, Guillermo; Kassen, Deepika; Williams, Andrew E; Mirza, Muddassar M; Balazs, Mercedesz; Wang, Xiaoting; Rodriguez, Robert Sanchez; Alendar, Andrej; Barker, Jonathan; Tsoka, Sophia; Ouyang, Wenjun; Nestle, Frank O

    2014-02-12

    Cytokines are critical checkpoints of inflammation. The treatment of human autoimmune disease has been revolutionized by targeting inflammatory cytokines as key drivers of disease pathogenesis. Despite this, there exist numerous pitfalls when translating preclinical data into the clinic. We developed an integrative biology approach combining human disease transcriptome data sets with clinically relevant in vivo models in an attempt to bridge this translational gap. We chose interleukin-22 (IL-22) as a model cytokine because of its potentially important proinflammatory role in epithelial tissues. Injection of IL-22 into normal human skin grafts produced marked inflammatory skin changes resembling human psoriasis. Injection of anti-IL-22 monoclonal antibody in a human xenotransplant model of psoriasis, developed specifically to test potential therapeutic candidates, efficiently blocked skin inflammation. Bioinformatic analysis integrating both the IL-22 and anti-IL-22 cytokine transcriptomes and mapping them onto a psoriasis disease gene coexpression network identified key cytokine-dependent hub genes. Using knockout mice and small-molecule blockade, we show that one of these hub genes, the so far unexplored serine/threonine kinase PIM1, is a critical checkpoint for human skin inflammation and potential future therapeutic target in psoriasis. Using in silico integration of human data sets and biological models, we were able to identify a new target in the treatment of psoriasis.

  20. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

    PubMed Central

    Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

    2011-01-01

    Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

  1. Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury.

    PubMed

    Lu, Naihao; Sui, Yinhua; Tian, Rong; Peng, Yi-Yuan

    2018-05-16

    Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

  2. Identifying the cellular targets of natural products using T7 phage display.

    PubMed

    Piggott, Andrew M; Karuso, Peter

    2016-05-04

    Covering: up to the end of 2015While Nature continues to deliver a myriad of potent and structurally diverse biologically active small molecules, the cellular targets and modes of action of these natural products are rarely identified, significantly hindering their development as new chemotherapeutic agents. This article provides an introductory tutorial on the use of T7 phage display as a tool to rapidly identify the cellular targets of natural products and is aimed specifically at natural products chemists who may have only limited experience in molecular biology. A brief overview of T7 phage display is provided, including its strengths, weaknesses, and the type of problems that can and cannot be tackled with this technology. Affinity probe construction is reviewed, including linker design and natural product derivatisation strategies. A detailed description of the T7 phage biopanning procedure is provided, with valuable tips for optimising each step in the process, as well as advice for identifying and avoiding the most commonly encountered challenges and pitfalls along the way. Finally, a brief discussion is provided on techniques for validating the cellular targets identified using T7 phage display.

  3. Genomes2Drugs: Identifies Target Proteins and Lead Drugs from Proteome Data

    PubMed Central

    Toomey, David; Hoppe, Heinrich C.; Brennan, Marian P.; Nolan, Kevin B.; Chubb, Anthony J.

    2009-01-01

    Background Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. Methodology/Principal Findings To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. Conclusions/Significance Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under ‘change-of-application’ patents. PMID:19593435

  4. Myeloperoxidase genotype, fruit and vegetable consumption, and breast cancer risk.

    PubMed

    Ahn, Jiyoung; Gammon, Marilie D; Santella, Regina M; Gaudet, Mia M; Britton, Julie A; Teitelbaum, Susan L; Terry, Mary Beth; Neugut, Alfred I; Josephy, P David; Ambrosone, Christine B

    2004-10-15

    Myeloperoxidase (MPO), an antimicrobial enzyme in the breast, generates reactive oxygen species (ROS) endogenously. An MPO G463A polymorphism exists in the promoter region, with the variant A allele conferring lower transcription activity than the common G allele. Because oxidative stress may play a role in breast carcinogenesis, we evaluated MPO genotypes in relation to breast cancer risk among 1,011 cases and 1,067 controls from the Long Island Breast Cancer Study Project (1996-1997). We also assessed the potential modifying effects of dietary antioxidants and hormonally related risk factors on these relationships. Women over 20 years with incident breast cancer who were residents of Nassau and Suffolk Counties, NY, were identified as potential cases. Population-based controls were frequency matched by 5-year age groups. Genotyping was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) technology, and suspected breast cancer risk factors and usual dietary intake were assessed during an in-person interview. Unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals. Having at least one A allele was associated with an overall 13% reduction in breast cancer risk. When consumption of fruits and vegetables and specific dietary antioxidants were dichotomized at the median, inverse associations with either GA or AA genotypes were most pronounced among women who consumed higher amounts of total fruits and vegetables (odds ratio, 0.75; 95% confidence interval, 0.58-0.97); this association was not noted among the low-consumption group (P for interaction = 0.04). Relationships were strongest among premenopausal women. Results from this first study of MPO genotypes and breast cancer risk indicate that MPO variants, related to reduced generation of ROS, are associated with decreased breast cancer risk, and emphasize the importance of fruit and vegetable consumption in reduction of breast

  5. Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

    PubMed

    Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

    2012-11-01

    The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

    PubMed

    Bian, Zhi-Ping; Li, Xiong-Zhi; Wu, Heng-Fang; Xu, Jin-Dan; Gu, Chun-Rong; Chen, Xiang-Jian; Yang, Di

    2016-04-01

    Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

  7. Myeloperoxidase potentiates nitric oxide-mediated nitrosation.

    PubMed

    Lakshmi, Vijaya M; Nauseef, William M; Zenser, Terry V

    2005-01-21

    Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 mum) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 mum H(2)O(2)/min, and 0.024 mum NO/min. Autoxidation was only detected at >/=1.2 mum NO/min. MPO potentiation was not affected by a 40-fold excess flux of H(2)O(2) over NO or less than a 2.4-fold excess flux of NO over H(2)O(2). Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO(+). MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO(2)(.) or an NO(2)(.)-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO(2)(-) at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO(2)(-). Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN(3). This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration.

  8. Combined expressional analysis, bioinformatics and targeted proteomics identify new potential therapeutic targets in glioblastoma stem cells.

    PubMed

    Stangeland, Biljana; Mughal, Awais A; Grieg, Zanina; Sandberg, Cecilie Jonsgar; Joel, Mrinal; Nygård, Ståle; Meling, Torstein; Murrell, Wayne; Vik Mo, Einar O; Langmoen, Iver A

    2015-09-22

    Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies.To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways.Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.

  9. Oxidative stress and myeloperoxidase levels in saliva of patients with recurrent aphthous stomatitis.

    PubMed

    Cağlayan, F; Miloglu, O; Altun, O; Erel, O; Yilmaz, A B

    2008-11-01

    Recurrent aphthous stomatitis (RAS) is the most common oral ulcerative condition affecting 5-25% of the general population. The aim of this study was to evaluate the oxidative stress parameters in saliva of patients with RAS and to investigate the relationship among these parameters in either group. The study involved 50 patients with RAS of whom 24 were male and 26 were female, and 25 healthy controls of whom 13 were male and 12 were female. There was no statistically significant difference in the salivary total antioxidant capacity, total oxidant status, oxidative stress index levels, and myeloperoxidase activity between patients with RAS and those in the control group. The results show that reactive oxygen species may not play a role in the etiology of RAS.

  10. Genome-wide and gene-centric analyses of circulating myeloperoxidase levels in the charge and care consortia.

    PubMed

    Reiner, Alexander P; Hartiala, Jaana; Zeller, Tanja; Bis, Joshua C; Dupuis, Josée; Fornage, Myriam; Baumert, Jens; Kleber, Marcus E; Wild, Philipp S; Baldus, Stephan; Bielinski, Suzette J; Fontes, João D; Illig, Thomas; Keating, Brendan J; Lange, Leslie A; Ojeda, Francisco; Müller-Nurasyid, Martina; Munzel, Thomas F; Psaty, Bruce M; Rice, Kenneth; Rotter, Jerome I; Schnabel, Renate B; Tang, W H Wilson; Thorand, Barbara; Erdmann, Jeanette; Jacobs, David R; Wilson, James G; Koenig, Wolfgang; Tracy, Russell P; Blankenberg, Stefan; März, Winfried; Gross, Myron D; Benjamin, Emelia J; Hazen, Stanley L; Allayee, Hooman

    2013-08-15

    Increased systemic levels of myeloperoxidase (MPO) are associated with the risk of coronary artery disease (CAD). To identify the genetic factors that are associated with circulating MPO levels, we carried out a genome-wide association study (GWAS) and a gene-centric analysis in subjects of European ancestry and African Americans (AAs). A locus on chromosome 1q31.1 containing the complement factor H (CFH) gene was strongly associated with serum MPO levels in 9305 subjects of European ancestry (lead SNP rs800292; P = 4.89 × 10(-41)) and in 1690 AA subjects (rs505102; P = 1.05 × 10(-8)). Gene-centric analyses in 8335 subjects of European ancestry additionally identified two rare MPO coding sequence variants that were associated with serum MPO levels (rs28730837, P = 5.21 × 10(-12); rs35897051, P = 3.32 × 10(-8)). A GWAS for plasma MPO levels in 9260 European ancestry subjects identified a chromosome 17q22 region near MPO that was significantly associated (lead SNP rs6503905; P = 2.94 × 10(-12)), but the CFH locus did not exhibit evidence of association with plasma MPO levels. Functional analyses revealed that rs800292 was associated with levels of complement proteins in serum. Variants at chromosome 17q22 also had pleiotropic cis effects on gene expression. In a case-control analysis of ∼80 000 subjects from CARDIoGRAM, none of the identified single-nucleotide polymorphisms (SNPs) were associated with CAD. These results suggest that distinct genetic factors regulate serum and plasma MPO levels, which may have relevance for various acute and chronic inflammatory disorders. The clinical implications for CAD and a better understanding of the functional basis for the association of CFH and MPO variants with circulating MPO levels require further study.

  11. How reliable an indicator of inflammation is myeloperoxidase activity?

    PubMed

    Faith, Minnie; Sukumaran, Abitha; Pulimood, Anna B; Jacob, Molly

    2008-10-01

    Myeloperoxidase (MPO) and interleukin-6 (IL-6) are often used as markers of inflammation. The aim of this study was to ascertain whether MPO activity is as reliable as IL-6 as an indicator of inflammation. Inflammation was induced in mice, using either turpentine or indomethacin. Duodenal tissue was removed from these animals at various time periods ranging from 6 h to 7 days later. Concentrations of IL-6 and MPO activity were estimated in the tissue. Histopathological examination was also carried out at some of the time periods to determine the presence of neutrophil infiltration in turpentine-treated mice. Concentrations of IL-6 and MPO activity were significantly higher in tissue that had been treated with the agents used, at all the time periods studied, when compared with corresponding control tissue. Fold-increases in MPO activity were higher than fold-increases in IL-6. Concentrations of the 2 parameters showed significant positive correlation. Histopathological examination did not show significantly higher numbers of neutrophils infiltrating the tissue in response to turpentine, at the time periods studied. Estimation of MPO activity is a reliable indicator of inflammation, being more sensitive than histopathological examination of tissue and as good as measurement of IL-6 concentrations.

  12. In vitro activation of dibromoacetonitrile to cyanide by myeloperoxidase.

    PubMed

    Al-Abbasi, Fahad A

    2016-08-01

    Dibromoacetonitrile (DBAN) is a disinfection by-product classified as a potential human and animal carcinogen. This study aimed at investigating the ability of myeloperoxidase (MPO) to oxidize DBAN to cyanide (CN - ) in vitro Detection of CN - served as a marker for the possible generation of free radical intermediates implicated in DBAN-induced toxicity. Optimum conditions for the oxidation of DBAN to CN - were characterized with respect to pH, temperature, and time of incubation as well as DBAN, MPO, potassium chloride, and hydrogen peroxide (H 2 O 2 ) concentrations in incubation mixtures. Maximum reaction velocity and Michaelis-Menten constant were assessed. Addition of sodium hypochlorite to the reaction mixtures significantly enhanced the rate of the reaction. Addition of the MPO inhibitors, sodium azide, 4-amino benzoic acid hydrazine, or indomethacin to the reaction mixtures significantly decreased the rate of DBAN oxidation. Inclusion of the antioxidant enzyme superoxide dismutase in the incubation mixtures significantly decreased the rate of reaction. Inclusion of the sulfhydryl compounds as reduced glutathione, N-acetylcysteine, d-penicillamine, or l-cysteine enhanced the rate of DBAN oxidation. These results demonstrate the ability of MPO/H 2 O 2 /chloride ion system to oxidize DBAN to CN - and provide insight for the elucidation of DBAN chronic toxicity. © The Author(s) 2015.

  13. Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets

    PubMed Central

    Akala, Hoseah M.; Macharia, Rosaline W.; Juma, Dennis W.; Cheruiyot, Agnes C.; Andagalu, Ben; Brown, Mathew L.; El-Shemy, Hany A.; Nyanjom, Steven G.

    2017-01-01

    Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens. PMID:29088219

  14. Identifying and targeting determinants of melanoma cellular invasion.

    PubMed

    Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L; Tan, BeeShin; Behren, Andreas; Cebon, Jonathan; McKeown, Sonja J

    2016-07-05

    Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

  15. Identifying and targeting determinants of melanoma cellular invasion

    PubMed Central

    Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L.; Tan, BeeShin

    2016-01-01

    Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients. PMID:27172792

  16. Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

    PubMed

    Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

    2018-05-22

    Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

  17. Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

    PubMed Central

    Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

    2017-01-01

    Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or

  18. Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

    PubMed Central

    Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

    2014-01-01

    MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

  19. Identifying Drug-Target Interactions with Decision Templates.

    PubMed

    Yan, Xiao-Ying; Zhang, Shao-Wu

    2018-01-01

    During the development process of new drugs, identification of the drug-target interactions wins primary concerns. However, the chemical or biological experiments bear the limitation in coverage as well as the huge cost of both time and money. Based on drug similarity and target similarity, chemogenomic methods can be able to predict potential drug-target interactions (DTIs) on a large scale and have no luxurious need about target structures or ligand entries. In order to reflect the cases that the drugs having variant structures interact with common targets and the targets having dissimilar sequences interact with same drugs. In addition, though several other similarity metrics have been developed to predict DTIs, the combination of multiple similarity metrics (especially heterogeneous similarities) is too naïve to sufficiently explore the multiple similarities. In this paper, based on Gene Ontology and pathway annotation, we introduce two novel target similarity metrics to address above issues. More importantly, we propose a more effective strategy via decision template to integrate multiple classifiers designed with multiple similarity metrics. In the scenarios that predict existing targets for new drugs and predict approved drugs for new protein targets, the results on the DTI benchmark datasets show that our target similarity metrics are able to enhance the predictive accuracies in two scenarios. And the elaborate fusion strategy of multiple classifiers has better predictive power than the naïve combination of multiple similarity metrics. Compared with other two state-of-the-art approaches on the four popular benchmark datasets of binary drug-target interactions, our method achieves the best results in terms of AUC and AUPR for predicting available targets for new drugs (S2), and predicting approved drugs for new protein targets (S3).These results demonstrate that our method can effectively predict the drug-target interactions. The software package can

  20. Identification of Tengfu Jiangya Tablet Target Biomarkers with Quantitative Proteomic Technique

    PubMed Central

    Xu, Jingwen; Zhang, Shijun; Jiang, Haiqiang; Wang, Nan; Lin, Haiqing

    2017-01-01

    Tengfu Jiangya Tablet (TJT) is a well accepted antihypertension drug in China and its major active components were Uncaria total alkaloids and Semen Raphani soluble alkaloid. To further explore treatment effects mechanism of TJT on essential hypertension, a serum proteomic study was performed. Potential biomarkers were quantified in serum of hypertension individuals before and after taking TJT with isobaric tags for relative and absolute quantitation (iTRAQ) coupled two-dimensional liquid chromatography followed electrospray ionization-tandem mass spectrometry (2D LC-MS/MS) proteomics technique. Among 391 identified proteins with high confidence, 70 proteins were differentially expressed (fold variation criteria, >1.2 or <0.83) between two groups (39 upregulated and 31 downregulated). Combining with Gene Ontology annotation, KEGG pathway analysis, and literature retrieval, 5 proteins were chosen as key target biomarkers during TJT therapeutic process. And the alteration profiles of these 5 proteins were verified by ELISA and Western Blot. Proteins Kininogen 1 and Keratin 1 are members of Kallikrein system, while Myeloperoxidase, Serum Amyloid protein A, and Retinol binding protein 4 had been reported closely related to vascular endothelial injury. Our study discovered 5 target biomarkers of the compound Chinese medicine TJT. Secondly, this research initially revealed the antihypertension therapeutic mechanism of this drug from a brand-new aspect. PMID:28408942

  1. Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

    PubMed

    Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

    2017-08-01

    Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

  2. Myeloperoxidase-Mediated Iodination by Granulocytes INTRACELLULAR SITE OF OPERATION AND SOME REGULATING FACTORS

    PubMed Central

    Root, Richard K.; Stossel, Thomas P.

    1974-01-01

    The intracellular site of operation of the myeloperoxidase-H2O2-halide antibacterial system of granulocytes has been determined by utilizing measurements of the fixation of iodide to trichloracetic acid (TCA) precipitates of subcellular fractions, including intact phagocytic vesicles. Na125I was added to suspensions of guinea pig granulocytes in Krebs-Ringer phosphate buffer, and they were then permitted to phagocytize different particles. Phagocytic vesicles were formed by allowing cells to ingest a paraffin oil emulsion (POE) and collected by flotation on sucrose after homogenization. Measurement of 125I bound to TCA precipitates of the different fractions and the homogenates disclosed that the lysosome-rich fraction obtained by centrifugation from control (nonphagocytizing) cells accounted for a mean 93.1% of the total cellular activity. With phagocytosis of POE, TCA-precipitable iodination increased two- to sevenfold, and the lysosomal contribution fell to a mean 36.9% of the total. The appearance of activity within phagocytic vesicles accounted for almost the entire increase seen with phagocytosis (a mean 75.7%), and iodide was bound within these structures with high specific activity. More iodide was taken up by cells than fixed, regardless of iodide concentration, and was distributed widely throughout the cell rather than selectively trapped within the vesicles. The amount of iodide taken up and fixed varied considerably with the phagocytic particle employed. Yeast particles were found to stimulate iodination to a far greater degree than the ingestion of POE or latex. Such observations are consistent with the concept that the ingested particle is a major recipient of the iodination process. Measurements of metabolic activities related to the formation and utilization of peroxide by cells phagocytizing different particles were made and correlated with iodination. The findings suggest that mechanisms must exist within granulocytes to collect or perhaps even

  3. Computational analysis of ribonomics datasets identifies long non-coding RNA targets of γ-herpesviral miRNAs.

    PubMed

    Sethuraman, Sunantha; Thomas, Merin; Gay, Lauren A; Renne, Rolf

    2018-05-29

    Ribonomics experiments involving crosslinking and immuno-precipitation (CLIP) of Ago proteins have expanded the understanding of the miRNA targetome of several organisms. These techniques, collectively referred to as CLIP-seq, have been applied to identifying the mRNA targets of miRNAs expressed by Kaposi's Sarcoma-associated herpes virus (KSHV) and Epstein-Barr virus (EBV). However, these studies focused on identifying only those RNA targets of KSHV and EBV miRNAs that are known to encode proteins. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are also targeted by miRNAs. In this study, we performed a systematic re-analysis of published datasets from KSHV- and EBV-driven cancers. We used CLIP-seq data from lymphoma cells or EBV-transformed B cells, and a crosslinking, ligation and sequencing of hybrids dataset from KSHV-infected endothelial cells, to identify novel lncRNA targets of viral miRNAs. Here, we catalog the lncRNA targetome of KSHV and EBV miRNAs, and provide a detailed in silico analysis of lncRNA-miRNA binding interactions. Viral miRNAs target several hundred lncRNAs, including a subset previously shown to be aberrantly expressed in human malignancies. In addition, we identified thousands of lncRNAs to be putative targets of human miRNAs, suggesting that miRNA-lncRNA interactions broadly contribute to the regulation of gene expression.

  4. Identifying mRNA sequence elements for target recognition by human Argonaute proteins

    PubMed Central

    Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

    2014-01-01

    It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

  5. Factor Analysis of Therapist-Identified Treatment Targets in Community-Based Children's Mental Health.

    PubMed

    Love, Allison R; Okado, Izumi; Orimoto, Trina E; Mueller, Charles W

    2018-01-01

    The present study used exploratory and confirmatory factor analyses to identify underlying latent factors affecting variation in community therapists' endorsement of treatment targets. As part of a statewide practice management program, therapist completed monthly reports of treatment targets (up to 10 per month) for a sample of youth (n = 790) receiving intensive in-home therapy. Nearly 75 % of youth were diagnosed with multiple co-occurring disorders. Five factors emerged: Disinhibition, Societal Rules Evasion, Social Engagement Deficits, Emotional Distress, and Management of Biodevelopmental Outcomes. Using logistic regression, primary diagnosis predicted therapist selection of Disinhibition and Emotional Distress targets. Client age predicted endorsement of Societal Rules Evasion targets. Practice-to-research implications are discussed.

  6. Inhibition of myeloperoxidase and antioxidative activity of Gentiana lutea extracts.

    PubMed

    Nastasijević, Branislav; Lazarević-Pašti, Tamara; Dimitrijević-Branković, Suzana; Pašti, Igor; Vujačić, Ana; Joksić, Gordana; Vasić, Vesna

    2012-07-01

    The aim of this study was to investigate the inhibitory activity of Gentiana lutea extracts on the enzyme myeloperoxidase (MPO), as well as the antioxidant activity of these extracts and their correlation with the total polyphenol content. Extracts were prepared using methanol (100%), water and ethanol aqueous solutions (96, 75, 50 and 25%v/v) as solvents for extraction. Also, isovitexin, amarogentin and gentiopicroside, pharmacologically active constituents of G. lutea were tested as potential inhibitors of MPO. Antioxidant activity of extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging test and also using cyclic voltammetry (CV). Among all extracts, the antioxidant capacity of 50% ethanol aqueous extract was the highest, both when measured using the DPPH test, with IC(50)=20.6 μg/ml, and when using CV. Also, 50% ethanol extract, showed the best inhibition of MPO activity in comparison with other extracts. In the group of the selected G. lutea constituents, gentiopicroside has proved to be the strongest inhibitor of MPO, with IC(50)=0.8 μg/ml. Also, the concentration of G. lutea constituents were determined in all extracts, using Ultra Performance Liquid Chromatography (UPLC). Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

    DOE PAGES

    Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

    2015-03-30

    Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

  8. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

    Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

  9. Regulation of the nitric oxide oxidase activity of myeloperoxidase by pharmacological agents.

    PubMed

    Maiocchi, Sophie L; Morris, Jonathan C; Rees, Martin D; Thomas, Shane R

    2017-07-01

    The leukocyte-derived heme enzyme myeloperoxidase (MPO) is released extracellularly during inflammation and impairs nitric oxide (NO) bioavailability by directly oxidizing NO or producing NO-consuming substrate radicals. Here, structurally diverse pharmacological agents with activities as MPO substrates/inhibitors or antioxidants were screened for their effects on MPO NO oxidase activity in human plasma and physiological model systems containing endogenous MPO substrates/antioxidants (tyrosine, urate, ascorbate). Hydrazide-based irreversible/reversible MPO inhibitors (4-ABAH, isoniazid) or the sickle cell anaemia drug, hydroxyurea, all promoted MPO NO oxidase activity. This involved the capacity of NO to antagonize MPO inhibition by hydrazide-derived radicals and/or the ability of drug-derived radicals to stimulate MPO turnover thereby increasing NO consumption by MPO redox intermediates or NO-consuming radicals. In contrast, the mechanism-based irreversible MPO inhibitor 2-thioxanthine, potently inhibited MPO turnover and NO consumption. Although the phenolics acetaminophen and resveratrol initially increased MPO turnover and NO consumption, they limited the overall extent of NO loss by rapidly depleting H 2 O 2 and promoting the formation of ascorbyl radicals, which inefficiently consume NO. The vitamin E analogue trolox inhibited MPO NO oxidase activity in ascorbate-depleted fluids by scavenging NO-consuming tyrosyl and urate radicals. Tempol and related nitroxides decreased NO consumption in ascorbate-replete fluids by scavenging MPO-derived ascorbyl radicals. Indoles or apocynin yielded marginal effects. Kinetic analyses rationalized differences in drug activities and identified criteria for the improved inhibition of MPO NO oxidase activity. This study reveals that widely used agents have important implications for MPO NO oxidase activity under physiological conditions, highlighting new pharmacological strategies for preserving NO bioavailability during

  10. Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I*

    PubMed Central

    Chan, Gary K. L.; Witkowski, Andrzej; Gantz, Donald L.; Zhang, Tianqi O.; Zanni, Martin T.; Jayaraman, Shobini; Cavigiolio, Giorgio

    2015-01-01

    High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo. PMID:25759391

  11. Identifying Belief-Based Targets for the Promotion of Leisure-Time Walking

    ERIC Educational Resources Information Center

    Rhodes, Ryan E.; Blanchard, Chris M.; Courneya, Kerry S.; Plotnikoff, Ronald C.

    2009-01-01

    Walking is the most common type of physical activity (PA) and the likely target of efforts to increase PA. No studies, however, have identified the belief-level correlates for walking using the theory of planned behavior. This study elicits salient beliefs about walking and evaluates beliefs that may be most important for walking-promotion…

  12. Myeloperoxidase Antineutrophil Cytoplasmic Antibody (MPO-ANCA) Associated Crescentic and Necrotizing Glomerulonephritis (GN) with Membranoproliferative GN Features.

    PubMed

    Koda, Ryo; Nagahori, Katsuhiro; Kitazawa, Atsushi; Imanishi, Yuji; Yoshino, Atsunori; Kawamoto, Shinya; Ueda, Yoshihiko; Takeda, Tetsuro

    2016-01-01

    A 77-year-old man presented with a fever, non-productive cough, and edema formation. A laboratory analysis showed an elevated creatinine level (2.5 mg/dL), a high titer of myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA) (99 U/mL), positive reaction for antinuclear antibody (×320), hematuria, and massive proteinuria (3.33 g/day). A renal biopsy revealed crescentic and necrotizing glomerulonephritis (GN) with membranoproliferative GN features [double contour appearance of the glomerular basement membrane, granular deposition of immunoglobulin (Ig) G, IgM, and C3 along the capillary wall, subendothelial and subepithelial deposits with mesangial interposition]. A potential relationship between MPO-ANCA associated GN and membranoproliferative GN is discussed.

  13. In silico molecular comparisons of C. elegans and mammalian pharmacology identify distinct targets that regulate feeding.

    PubMed

    Lemieux, George A; Keiser, Michael J; Sassano, Maria F; Laggner, Christian; Mayer, Fahima; Bainton, Roland J; Werb, Zena; Roth, Bryan L; Shoichet, Brian K; Ashrafi, Kaveh

    2013-11-01

    Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

  14. Novel target for high-risk neuroblastoma identified in pre-clinical research | Center for Cancer Research

    Cancer.gov

    Pre-clinical research by investigators at the Center for Cancer Research and their colleagues have identified a number of novel epigenetic targets for high-risk neuroblastoma and validated a promising new targeted inhibitor in pre-clinical models.  Read more...

  15. GRIL-Seq, a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

    PubMed Central

    Han, Kook; Tjaden, Brian; Lory, Stephen

    2017-01-01

    The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but can also result in uncovering novel roles for sRNAs and their targets in complex regulatory networks. PMID:28005055

  16. Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes.

    PubMed Central

    Capeillère-Blandin, C; Martin, C; Gaggero, N; Pasta, P; Carrea, G; Colonna, S

    1998-01-01

    The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 degreesC. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1-10 s-1 range and around 43+/-26 microM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (sigma+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4-8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3-4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity. PMID:9742209

  17. Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes.

    PubMed

    Capeillère-Blandin, C; Martin, C; Gaggero, N; Pasta, P; Carrea, G; Colonna, S

    1998-10-01

    The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 degreesC. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1-10 s-1 range and around 43+/-26 microM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (sigma+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4-8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3-4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity.

  18. The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

    PubMed Central

    Lane, Amanda E.; Tan, Joanne T. M.; Hawkins, Clare L.; Heather, Alison K.; Davies, Michael J.

    2010-01-01

    MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by hydrogen peroxide to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Specificity constants indicate that SCN− is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. While HOCl and HOBr are powerful oxidizing agents, HOSCN is a less reactive, but more specific, oxidant which targets thiols and especially low pKa species. In the present study we show that HOSCN targets cysteine residues present in PTPs (protein tyrosine phosphatases) with this resulting in a loss of PTP activity for the isolated enzyme, in cell lysates and intact J774A.1 macrophage-like cells. Inhibition also occurs with MPO-generated HOCl and HOBr, but is more marked with MPO-generated HOSCN, particularly at longer incubation times. This inhibition is reversed by dithiothreitol, particularly at early time points, consistent with the reversible oxidation of the active site cysteine residue to give either a cysteine–SCN adduct or a sulfenic acid. Inhibition of PTP activity is associated with increased phosphorylation of p38a and ERK2 (extracellular-signal-regulated kinase 2) as detected by Western blot analysis and phosphoprotein arrays, and results in altered MAPK (mitogen-activated protein kinase) signalling. These data indicate that the highly selective targeting of some protein thiols by HOSCN can result in perturbation of cellular phosphorylation and altered cell signalling. These changes occur with (patho)physiological concentrations of SCN− ions, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage, particularly in smokers who have elevated plasma levels of SCN−. PMID:20528774

  19. Myeloperoxidase scavenges peroxynitrite: A novel anti-inflammatory action of the heme enzyme

    PubMed Central

    Koyani, Chintan N.; Flemmig, Joerg; Malle, Ernst; Arnhold, Juergen

    2015-01-01

    Peroxynitrite, a potent pro-inflammatory and cytotoxic species, interacts with a variety of heme containing proteins. We addressed the question whether (i) the interaction of myeloperoxidase (MPO, an enzyme generating hypochlorous acid from hydrogen peroxide and chloride ions) with peroxynitrite affects the clearance of peroxynitrite, and (ii) if peroxynitrite could modulate the chlorinating activity of MPO. Our results show that this interaction promotes the decomposition of the highly reactive pro-inflammatory oxidant, whereby MPO Compound II (but not Compound I) is formed. The efficiency of MPO to remove peroxynitrite was enhanced by l-tyrosine, nitrite and (−)-epicatechin, substances known to reduce Compound II with high reaction rate. Next, peroxynitrite (added as reagent) diminished the chlorinating activity of MPO in the presence of hydrogen peroxide. Alternatively, SIN-1, a peroxynitrite donor, reduced hypochlorous acid formation by MPO, as measured by aminophenyl fluorescein oxidation (time kinetics) and taurine chloramine formation (end point measurement). At inflammatory loci, scavenging of peroxynitrite by MPO may overcome the uncontrolled peroxynitrite decomposition and formation of reactive species, which lead to cell/tissue damage. PMID:25731855

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

    PubMed Central

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

    2012-01-01

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

  1. Combining functional genomics and chemical biology to identify targets of bioactive compounds.

    PubMed

    Ho, Cheuk Hei; Piotrowski, Jeff; Dixon, Scott J; Baryshnikova, Anastasia; Costanzo, Michael; Boone, Charles

    2011-02-01

    Genome sequencing projects have revealed thousands of suspected genes, challenging researchers to develop efficient large-scale functional analysis methodologies. Determining the function of a gene product generally requires a means to alter its function. Genetically tractable model organisms have been widely exploited for the isolation and characterization of activating and inactivating mutations in genes encoding proteins of interest. Chemical genetics represents a complementary approach involving the use of small molecules capable of either inactivating or activating their targets. Saccharomyces cerevisiae has been an important test bed for the development and application of chemical genomic assays aimed at identifying targets and modes of action of known and uncharacterized compounds. Here we review yeast chemical genomic assays strategies for drug target identification. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

    PubMed Central

    Azorsa, David O; Gonzales, Irma M; Basu, Gargi D; Choudhary, Ashish; Arora, Shilpi; Bisanz, Kristen M; Kiefer, Jeffrey A; Henderson, Meredith C; Trent, Jeffrey M; Von Hoff, Daniel D; Mousses, Spyro

    2009-01-01

    Background Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. Methods In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. Results Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. Conclusion These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine. PMID

  3. GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation.

    PubMed

    Han, Kook; Tjaden, Brian; Lory, Stephen

    2016-12-22

    The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

  4. Distinct gene alterations with a high percentage of myeloperoxidase-positive leukemic blasts in de novo acute myeloid leukemia.

    PubMed

    Kamijo, Rena; Itonaga, Hidehiro; Kihara, Rika; Nagata, Yasunobu; Hata, Tomoko; Asou, Norio; Ohtake, Shigeki; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Ogawa, Seishi; Naoe, Tomoki; Kiyoi, Hitoshi; Miyazaki, Yasushi

    2018-02-01

    The myeloperoxidase (MPO)-positivity of blasts in bone marrow smears is an important marker for not only the diagnosis, but also the prognosis of acute myeloid leukemia (AML). To investigate the relationship between genetic alterations and MPO-positivity, we performed targeted sequencing for 51 genes and 10 chimeric gene transcripts in 164 newly diagnosed de novo AML patients; 107 and 57 patients were classified as AML with >50% MPO-positive blasts (MPO-high group) and ≤50% MPO-positive blasts, (MPO-low group), respectively. The univariate analysis revealed that RUNX1-RUNX1T1 (P < 0.001), the KIT mutation (P < 0.001), and CEBPA double mutation (P = 0.001) were more likely to be found in the MPO-high group, while the DNMT3A mutation (P = 0.001), FLT3 tyrosine kinase domain mutation (P = 0.004), and TP53 mutation (P = 0.020) were more likely to be present in the MPO-low group. Mutations in genes related to DNA hypermethylation signatures (IDH1, IDH2, TET2, and WT1 genes) were more frequent in the MPO-high group (P = 0.001) when patients with fusion genes of core-binding factors were excluded from the analysis. Our results suggest that MPO-positivity of blasts was related with the distinct gene mutation patterns among de novo AML patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Omen: identifying potential spear-phishing targets before the email is sent.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wendt, Jeremy Daniel.

    2013-07-01

    We present the results of a two year project focused on a common social engineering attack method called "spear phishing". In a spear phishing attack, the user receives an email with information specifically focused on the user. This email contains either a malware-laced attachment or a link to download the malware that has been disguised as a useful program. Spear phishing attacks have been one of the most effective avenues for attackers to gain initial entry into a target network. This project focused on a proactive approach to spear phishing. To create an effective, user-specific spear phishing email, the attackermore » must research the intended recipient. We believe that much of the information used by the attacker is provided by the target organization's own external website. Thus when researching potential targets, the attacker leaves signs of his research in the webserver's logs. We created tools and visualizations to improve cybersecurity analysts' abilities to quickly understand a visitor's visit patterns and interests. Given these suspicious visitors and log-parsing tools, analysts can more quickly identify truly suspicious visitors, search for potential spear-phishing targeted users, and improve security around those users before the spear phishing email is sent.« less

  6. Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

    PubMed Central

    Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

    2015-01-01

    Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

  7. Oxidant-induced DNA damage of target cells.

    PubMed Central

    Schraufstätter, I; Hyslop, P A; Jackson, J H; Cochrane, C G

    1988-01-01

    In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks. PMID:2843565

  8. A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18*

    PubMed Central

    Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

    2017-01-01

    Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. PMID:28087594

  9. Targeted Approach to Identify Genetic Loci Associated with ...

    EPA Pesticide Factsheets

    Extreme tolerance to highly toxic dioxin-like contaminants (DLCs) has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly contaminated urban/industrialized estuaries of the US Atlantic coast. We hypothesized that comparisons among tolerant populations and in contrast to their sensitive neighboring killifish might reveal genetic loci associated with DLC tolerance. Since the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, we identified single nucleotide polymorphisms (SNPs) from 43 genes associated with the AHR to serve as targeted markers. Wild fish from the four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Consistent with other killifish population genetic analyses, our results revealed strong genetic differentiation among populations, consistent with isolation by distance models. Pairwise comparisons of nearby tolerant and sensitive populations revealed differentiation among these loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP) 1A and 3A30, and the NADH ubiquinone oxidoreductase MLRQ subunit. By grouping tolerant versus sensitive populations, we also identified cytochrome P450 1A and the AHR2 loci as under selection, lend

  10. Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer

    PubMed Central

    Kim, Yunee; Jeon, Jouhyun; Mejia, Salvador; Yao, Cindy Q; Ignatchenko, Vladimir; Nyalwidhe, Julius O; Gramolini, Anthony O; Lance, Raymond S; Troyer, Dean A; Drake, Richard R; Boutros, Paul C; Semmes, O. John; Kislinger, Thomas

    2016-01-01

    Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers. PMID:27350604

  11. [The significances of peripheral neutrophils CD(55) and myeloperoxidase expression in patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody associated vasculitis].

    PubMed

    Zhou, X L; Zheng, M J; Shuai, Z W; Zhang, L; Zhang, M M; Chen, S Y

    2017-06-01

    Objective: To investigate the expression of CD(55) and myeloperoxidase (MPO) on neutrophils in patients with MPO-specific anti-neutrophil cytoplasmic antibody associated vasculitis(MPO-AAV), and analyze the relationship between the expression and clinical manifestation. Methods: Forty untreated patients with active MPO-AAV (patient group) and 30 healthy volunteers (control group) were enrolled in this study. The CD(55) on neutrophils and both membrane and cytoplasmic MPO were detected by flow cytometry. Serum fragment-from the activated complement factor B(Ba) and MPO were measured by ELISA. The clinical activity of vasculitis was valued by Birmingham vasculitis activity score-version 3(BVAS-V3). The significance of laboratory data was evaluated by Spearman correlation test and multivariate linear regression analysis. Results: (1)The mean fluorescence intensity(MFI) of CD(55) expressed on neutrophils was significantly higher than that in control group[4 068.6±2 306.0 vs 2 999.5±1 504.9, P =0.033]. Similar results of serum MPO and Ba in patient group were found compared to controls [500.0(381.0, 612.7) IU/L vs 286.9(225.5, 329.1) IU/L, P <0.001; 35.2(25.2, 79.5) ng/L vs 18.0(15.0, 28.0) ng/L, P <0.001], respectively. However, MIF of cytoplasmic MPO in patients was significantly lower than that of control group(1 577.1±1 175.9 vs 3 105.3±2 323.0, P =0.003) . (2) In patient group, cytoplasmic intensity of MPO was negatively associated with the serum levels of MPO( r =-0.710, P <0.001) and Ba ( r =-0.589, P =0.001). Moreover, serum MPO was positively associated with serum Ba( r =0.691, P <0.001). Membrane intensity of CD(55) on neutrophils was positively correlated with patient age ( r =0.514, P =0.001), C reactive protein ( r =0.376, P =0.018), peripheral neutrophils count ( r =0.485, P =0.001) and BVAS-V3 ( r =0.484, P =0.002), whereas negative correlation between membrane CD(55) and disease duration was seen ( r =-0.403, P =0.01). (3) The result of multiple

  12. Tyrosine 192 in apolipoprotein A-I is the major site of nitration and chlorination by myeloperoxidase, but only chlorination markedly impairs ABCA1-dependent cholesterol transport.

    PubMed

    Shao, Baohai; Bergt, Constanze; Fu, Xiaoyun; Green, Pattie; Voss, John C; Oda, Michael N; Oram, John F; Heinecke, Jay W

    2005-02-18

    High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO(-)). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO(-). Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr(192) of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO(-), suggesting that solvent accessibility accounted in part for the reactivity of Tyr(192). The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) is the predominant site of nitration and chlorination when MPO or ONOO(-) oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.

  13. Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

    PubMed

    Aroonsri, Aiyada; Akinola, Olugbenga; Posayapisit, Navaporn; Songsungthong, Warangkhana; Uthaipibull, Chairat; Kamchonwongpaisan, Sumalee; Gbotosho, Grace O; Yuthavong, Yongyuth; Shaw, Philip J

    2016-07-01

    The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  14. RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

    PubMed

    Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O

    2010-08-18

    Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

  15. Emerging preclinical pharmacological targets for Parkinson's disease

    PubMed Central

    More, Sandeep Vasant; Choi, Dong-Kug

    2016-01-01

    Parkinson's disease (PD) is a progressive neurological condition caused by the degeneration of dopaminergic neurons in the basal ganglia. It is the most prevalent form of Parkinsonism, categorized by cardinal features such as bradykinesia, rigidity, tremors, and postural instability. Due to the multicentric pathology of PD involving inflammation, oxidative stress, excitotoxicity, apoptosis, and protein aggregation, it has become difficult to pin-point a single therapeutic target and evaluate its potential application. Currently available drugs for treating PD provide only symptomatic relief and do not decrease or avert disease progression resulting in poor patient satisfaction and compliance. Significant amount of understanding concerning the pathophysiology of PD has offered a range of potential targets for PD. Several emerging targets including AAV-hAADC gene therapy, phosphodiesterase-4, potassium channels, myeloperoxidase, acetylcholinesterase, MAO-B, dopamine, A2A, mGlu5, and 5-HT-1A/1B receptors are in different stages of clinical development. Additionally, alternative interventions such as deep brain stimulation, thalamotomy, transcranial magnetic stimulation, and gamma knife surgery, are also being developed for patients with advanced PD. As much as these therapeutic targets hold potential to delay the onset and reverse the disease, more targets and alternative interventions need to be examined in different stages of PD. In this review, we discuss various emerging preclinical pharmacological targets that may serve as a new promising neuroprotective strategy that could actually help alleviate PD and its symptoms. PMID:26988916

  16. A screen to identify drug resistant variants to target-directed anti-cancer agents

    PubMed Central

    Azam, Mohammad; Raz, Tal; Nardi, Valentina; Opitz, Sarah L.

    2003-01-01

    The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia (CML)-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair. PMID:14615817

  17. Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

    DTIC Science & Technology

    2015-09-01

    assessed the specificity of mutation in Drosophila S2R+ cells. We generated a quantitative mutation reporter vector in which an sgRNA target sequence ...phosphatases (563 genes) in the Drosophila genome (Figure 4). 65 samples that displayed synthetic lethality (15 genes) or synthetic increases in viability...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . . Identified three hits (mRNA-Cap, Pitslre and CycT) that scored as

  18. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

    PubMed

    Manguso, Robert T; Pope, Hans W; Zimmer, Margaret D; Brown, Flavian D; Yates, Kathleen B; Miller, Brian C; Collins, Natalie B; Bi, Kevin; LaFleur, Martin W; Juneja, Vikram R; Weiss, Sarah A; Lo, Jennifer; Fisher, David E; Miao, Diana; Van Allen, Eliezer; Root, David E; Sharpe, Arlene H; Doench, John G; Haining, W Nicholas

    2017-07-27

    Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

  19. Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

    PubMed

    Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

    2016-11-21

    In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

  20. Researchers identify potential therapeutic targets for a rare childhood cancer | Center for Cancer Research

    Cancer.gov

    CCR researchers have identified the mechanism behind a rare but extremely aggressive childhood cancer called alveolar rhabdomyosarcoma (ARMS) and have pinpointed a potential drug target for its treatment. Learn more...

  1. Cellular myeloperoxidase activity in human monocytes stimulated by hyposialylated immunoglobulins and rheumatoid factors.

    PubMed Central

    Dodon, M D; Gazzolo, L; Quash, G A

    1984-01-01

    When hyposialylated , immunoglobulins become immunogenic and tend to form aggregates. In pursuit of the possibility that hyposialylated immunoglobulins (hs-Ig) can trigger human mononuclear phagocytic cells, we have investigated the effects of such hs-Ig on the myeloperoxidase (MPO) activity of these cells. The incubation of human monocytes with aggregated hs-Ig leads to the decrease of intracellular MPO activity. This decrease is dependent on the incubation time, on the amount of hs-Ig added, and on the degree of aggregation. Incubation with unaggregated hs-Ig has a similar effect, thus providing evidence that the loss of sialic acid residues per se is enough to render these molecules capable of decreasing the MPO content of phagocytic cells. Furthermore, human rheumatoid factors, isolated from the sera of rheumatoid arthritis patients, and previously characterized as hyposailylated Ig, interact in the same way with monocytes in triggering the MPO decrease. These observations imply that hs-Ig may be considered as active stimuli in the induction of inflammatory processes, through the initiation of oxidative reactions. PMID:6329948

  2. Assessment of Myeloperoxidase Activity by the Conversion of Hydroethidine to 2-Chloroethidium*

    PubMed Central

    Maghzal, Ghassan J.; Cergol, Katie M.; Shengule, Sudhir R.; Suarna, Cacang; Newington, Darren; Kettle, Anthony J.; Payne, Richard J.; Stocker, Roland

    2014-01-01

    Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, 1H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E+) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E+ was estimated to be 1.5 × 105 m−1s−1. To investigate the utility of 2-Cl-E+ to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo−/−) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E+ and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E+ and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E+ was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E+ were decreased substantially in Mpo−/− mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E+ was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E+ may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr. PMID:24436331

  3. A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo.

    PubMed

    Shazeeb, Mohammed S; Xie, Yang; Gupta, Suresh; Bogdanov, Alexei A

    2012-01-01

    Bis-phenylamides and bis-hydroxyindolamides of diethylenetriaminepentaacetic acid-gadolinium (DTPA(Gd)) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, gadolinium chelates of bis-5-hydroxytryptamide-DTPA (bis-5HT-DTPA(Gd)) have been used to image localized inflammation in animal models by detecting neutrophil-derived myeloperoxidase (MPO) activity at the inflammation site. However, in other preclinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here we report a novel MPO-sensing probe obtained by replacing the reducing substrate serotonin (5-HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using nuclear magnetic resonance spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd) (1) improves solubility in water; (2) acts as a substrate for both horseradish peroxidase and MPO enzymes; (3) induces cross-linking of proteins in the presence of MPO; (4) produces oxidation products, which bind to plasma proteins; and (5) unlike bis-5HT-DTPA(Gd), does not follow first-order reaction kinetics. In vivo magnetic resonance imaging (MRI) in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to 5 days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. Bis-HTrp-DTPA(Gd) should offer improvements for MRI of MPO-mediated inflammation in vivo, especially in high-field MRI, which requires a higher dose of contrast agent.

  4. A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo

    PubMed Central

    Shazeeb, Mohammed S.; Xie, Yang; Gupta, Suresh; Bogdanov, Alexei A.

    2013-01-01

    Bis-phenylamides and bis-hydroxyindolamides of DTPA(Gd) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, bis-5HT-DTPA(Gd) has been used to image localized inflammation in animal models by detecting neutrophil derived myeloperoxidase (MPO) activity at the inflammation site. However, in other pre-clinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here, we report a novel MPO sensing probe obtained by replacing the reducing substrate serotonin (5HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using NMR spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd): 1) improves solubility in water; 2) acts as a substrate for both HRP and MPO enzymes; 3) induces cross linking of proteins in the presence of MPO; 4) produces oxidation products which bind to plasma proteins and; 5) unlike bis-5HT-DTPA(Gd), does not follow first order reaction kinetics. In vivo MR imaging in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to five days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. In conclusion, bis-HTrp-DTPA(Gd) should offer improvements for MR imaging of MPO-mediated inflammation in vivo especially in high-field MRI, which requires higher dose of contrast agent. PMID:22954188

  5. Chemical proteomics approaches for identifying the cellular targets of natural products

    PubMed Central

    Sieber, S. A.

    2016-01-01

    Covering: 2010 up to 2016 Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied “in situ” – in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss ‘competitive mode’ approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed. PMID:27098809

  6. Chemical proteomics approaches for identifying the cellular targets of natural products.

    PubMed

    Wright, M H; Sieber, S A

    2016-05-04

    Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.

  7. Evaluation of bone marrow specimens with acute myelogenous leukemia for CD34, CD15, CD117, and myeloperoxidase.

    PubMed

    Dunphy, C H; Polski, J M; Evans, H L; Gardner, L J

    2001-08-01

    Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques. To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques. Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO). Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0. CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.

  8. Myeloperoxidase-catalyzed incorporation of amines into proteins: role of hypochlorous acid and dichloramines.

    PubMed

    Thomas, E L; Jefferson, M M; Grisham, M B

    1982-11-23

    Myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCl) resulted in formation of mono- and dichloramine derivatives (RNHCl and RNCl2) of primary amines. The RNCl2 derivatives could undergo a reaction that resulted in incorporation of the R moiety into proteins. The probable mechanism was attack of RNCl2 or an intermediate formed in the decomposition of RNCl2 on histidine, tyrosine, and cystine residues and on lysine residues at high pH. Incorporation of radioactivity from labeled amines into stable, high molecular weight derivatives of proteins was measured by acid or acetone precipitation and by gel chromatography and electrophoresis. Whereas formation of RNCl2 was favored at low pH, the subsequent incorporation reaction was favored at high pH. Up to several hours were required for the maximum amount of incorporation, which was less than 10% of the label in RNCl2. For the amines tested, incorporation was in the order histamine greater than 1,2-diaminoethane greater than putrescine greater than taurine greater than lysine greater than glucosamine greater than leucine greater than methylamine. Initiation of the reaction required HOCl, and oxidized forms of bromide, iodide, or thiocyanate did not substitute. Inhibitors of incorporation fell into three classes. First, ammonia or amines competed with the labeled amine for reaction with HOCl, so that larger amounts of HOCl were required. Second, readily oxidized substances such as sulfhydryl or diketo compounds or thioethers (methionine) reduced RNCl2. Third, certain compounds competed with protein as the acceptor for the incorporation reaction. The amount required to block incorporation into protein depended on protein concentration. Among these inhibitors were imidazole compounds (histidine), phenols (tyrosine), and disulfides (glutathione disulfide, GSSG). Low yields of derivatives of histidine, tyrosine, and GSSG were detected by thin-layer chromatography. Acid-precipitable derivatives were

  9. A proteomic approach to identifying new drug targets (potentiating topoisomerase II poisons).

    PubMed

    Jenkins, J R

    2008-10-01

    Topoisomerase II poisons are an established part of best clinical practice for the treatment of a number of solid tumours and haematological malignancies. However, toxicity and resistance to chemotherapeutic drugs often complicate the treatment. Furthermore, topoisomerase II poisons can also induce sister chromatid exchange, chromosomal recombination and chromosome aberrations and are associated with a significant risk of secondary leukaemia. It would therefore be of great clinical benefit if the efficacy of topoisomerase II inhibitors could be enhanced without the increased toxic side effects. It is proposed that clinical agents targeting topoisomerase II can be enhanced by inhibiting proteins that modulate topoisomerase II. The aim is to identify proteins, that by the nature of their interaction with topoisomerase II, represent putative drug targets.

  10. Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

    PubMed

    Yogalingam, Gouri; Lee, Amanda R; Mackenzie, Donald S; Maures, Travis J; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y; Hague, Chuck; Christianson, Terri; Bell, Sean M; LeBowitz, Jonathan H

    2017-03-10

    Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells*

    PubMed Central

    Yogalingam, Gouri; Lee, Amanda R.; Mackenzie, Donald S.; Maures, Travis J.; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y.; Hague, Chuck; Christianson, Terri; Bell, Sean M.; LeBowitz, Jonathan H.

    2017-01-01

    Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. PMID:28115520

  12. Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef).

    PubMed

    Cannarozzi, Gina; Plaza-Wüthrich, Sonia; Esfeld, Korinna; Larti, Stéphanie; Wilson, Yi Song; Girma, Dejene; de Castro, Edouard; Chanyalew, Solomon; Blösch, Regula; Farinelli, Laurent; Lyons, Eric; Schneider, Michel; Falquet, Laurent; Kuhlemeier, Cris; Assefa, Kebebew; Tadele, Zerihun

    2014-07-09

    Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

  13. PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3' UTRs and coding sequences.

    PubMed

    Šulc, Miroslav; Marín, Ray M; Robins, Harlan S; Vaníček, Jiří

    2015-07-01

    The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3' untranslated regions (3' UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3' UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA-mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA-mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Smart imaging of acute lung injury: exploration of myeloperoxidase activity using in vivo endoscopic confocal fluorescence microscopy.

    PubMed

    Chagnon, Frédéric; Bourgouin, Alexandra; Lebel, Réjean; Bonin, Marc-André; Marsault, Eric; Lepage, Martin; Lesur, Olivier

    2015-09-15

    The pathophysiology of acute lung injury (ALI) is well characterized, but its real-time assessment at bedside remains a challenge. When patients do not improve after 1 wk despite supportive therapies, physicians have to consider open lung biopsy (OLB) to identify the process(es) at play. Sustained inflammation and inadequate repair are often observed in this context. OLB is neither easy to perform in a critical setting nor exempt from complications. Herein, we explore intravital endoscopic confocal fluorescence microscopy (ECFM) of the lung in vivo combined with the use of fluorescent smart probe(s) activated by myeloperoxidase (MPO). MPO is a granular enzyme expressed by polymorphonuclear neutrophils (PMNs) and alveolar macrophages (AMs), catalyzing the synthesis of hypoclorous acid, a by-product of hydrogen peroxide. Activation of these probes was first validated in vitro in relevant cells (i.e., AMs and PMNs) and on MPO-non-expressing cells (as negative controls) and then tested in vivo using three rat models of ALI and real-time intravital imaging with ECFM. Semiquantitative image analyses revealed that in vivo probe-related cellular/background fluorescence was associated with corresponding enhanced lung enzymatic activity and was partly prevented by specific MPO inhibition. Additional ex vivo phenotyping was performed, confirming that fluorescent cells were neutrophil elastase(+) (PMNs) or CD68(+) (AMs). This work is a first step toward "virtual biopsy" of ALI without OLB. Copyright © 2015 the American Physiological Society.

  15. Silent genetic alterations identified by targeted next-generation sequencing in pheochromocytoma/paraganglioma: A clinicopathological correlations.

    PubMed

    Pillai, Suja; Gopalan, Vinod; Lo, Chung Y; Liew, Victor; Smith, Robert A; Lam, Alfred King Y

    2017-02-01

    The goal of this pilot study was to develop a customized, cost-effective amplicon panel (Ampliseq) for target sequencing in a cohort of patients with sporadic phaeochromocytoma/paraganglioma. Phaeochromocytoma/paragangliomas from 25 patients were analysed by targeted next-generation sequencing approach using an Ion Torrent PGM instrument. Primers for 15 target genes (NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, MEN1, KIF1Bβ, EPAS1, CDKN2 & PHD2) were designed using ion ampliseq designer. Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems were used to analysis these results. Overall, 713 variants were identified. The variants identified from the Ion Reporter ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 of these 713variants were deletions. Of these, six variants were non-pathogenic and four were likely to be pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Novel KIF1B pathogenic variant c.3375+1G>A was identified. The mutation was noted in a patient with clinically confirmed neurofibromatosis. Chromosome 1 showed the presence of maximum number of variants. Use of targeted next-generation sequencing is a sensitive method for the detecting genetic changes in patients with phaeochromocytoma/paraganglioma. The precise detection of these genetic changes helps in understanding the pathogenesis of these tumours. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

    PubMed Central

    Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

    2018-01-01

    The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

  17. Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets.

    PubMed

    Dey-Rao, Rama; Sinha, Animesh A

    2017-01-28

    Significant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state. We undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy. Unsupervised clustering methods of the VL-blood dataset demonstrate a "disease-state"-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as "hidden" (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional "hot spot" that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets. We examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address

  18. Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

    PubMed

    Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

    2015-10-01

    Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

    PubMed Central

    Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

    2012-01-01

    Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

  20. Red blood cells serve as intravascular carriers of myeloperoxidase.

    PubMed

    Adam, Matti; Gajdova, Silvie; Kolarova, Hana; Kubala, Lukas; Lau, Denise; Geisler, Anne; Ravekes, Thorben; Rudolph, Volker; Tsao, Philip S; Blankenberg, Stefan; Baldus, Stephan; Klinke, Anna

    2014-09-01

    Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. The Near-Earth Object Human Space Flight Accessible Targets Study (NHATS) List of Near-Earth Asteroids: Identifying Potential Targets for Future Exploration

    NASA Technical Reports Server (NTRS)

    Abell, Paul A.; Barbee, B. W.; Mink, R. G.; Alberding, C. M.; Adamo, D. R.; Mazanek, D. D.; Johnson, L. N.; Yeomans, D. K.; Chodas, P. W.; Chamberlin, A. B.; hide

    2012-01-01

    Over the past several years, much attention has been focused on the human exploration of near-Earth asteroids (NEAs). Two independent NASA studies examined the feasibility of sending piloted missions to NEAs [1, 2], and in 2009, the Augustine Commission identified NEAs as high profile destinations for human exploration missions beyond the Earth-Moon system [3]. More recently the current U.S. presidential administration directed NASA to include NEAs as destinations for future human exploration with the goal of sending astronauts to a NEA in the mid to late 2020s. This directive became part of the official National Space Policy of the United States of America as of June 28, 2010 [4]. Detailed planning for such deep space exploration missions and identifying potential NEAs as targets for human spaceflight requires selecting objects from the ever growing list of newly discovered NEAs. Hence NASA developed and implemented the Near-Earth Object (NEO) Human Space Flight (HSF) Accessible Target Study (NHATS), which identifies potential candidate objects on the basis of defined dynamical trajectory performance constraints.

  2. Hippocampal neurodegeneration in experimental autoimmune encephalomyelitis (EAE): potential role of inflammation activated myeloperoxidase.

    PubMed

    Sajad, Mir; Zargan, Jamil; Chawla, Raman; Umar, Sadiq; Sadaqat, Mir; Khan, Haider A

    2009-08-01

    Experimental Autoimmune Encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). The effect of this inflammatory disease on hippocampus has not been addressed. Keeping in view the above consideration an attempt was made to delineate the effect of EAE on the hippocampus of Wistar rats. The assessment of the damage to the hippocampus was done 16 days post induction by the immunolocalization of ChAT (choline acetyl transferase). ChAT decreased remarkably after induction that revealed cholinergic neuronal degeneration in the hippocampus. Subsequently, many biochemical parameters were assessed to ascertain inflammatory activation of nitric oxide and associated oxidative damage as a putative mechanism of the cholinergic degeneration. Nitric oxide metabolites increased significantly (P < 0.05) with enhancement of MPO (Myeloperoxidase activity) (P < 0.001) in the MOG (myelin oligodendrocyte protein) group as compared to the controls. Peroxidation of biomembranes increased (P < 0.001), while reduced glutathione depleted (P < 0.001) with parallel decrease in catalase (P < 0.01) and superoxide dismutase enzyme activity (P < 0.001) in the MOG group. Our results show a strong role of peroxidase dependent oxidation of nitrite and oxidative stress in cholinergic degeneration in EAE.

  3. Pre-separation storage of whole blood: the effect of temperature on red cell 2,3-diphosphoglycerate and myeloperoxidase in plasma.

    PubMed

    Knutson, F; Lööf, H; Högman, C F

    1999-10-01

    Although whole blood intended for component preparation is commonly left to cool at ambient temperature, knowledge is insufficient concerning what effects this may have on red blood cell (RBC) quality, in particular after a prolonged hold. Whole blood collected in CPD was incubated at 20 degrees C and 28 degrees C for 6 h designed as a paired study. Blood components were prepared and the red blood cell concentrates (RBCs) were stored for 28 days at 4 degrees C +/- 2 degrees C. Blood gases, pH, glucose, lactate, adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG) and plasma myeloperoxidase (MPO) were investigated. After 6 h the 2,3-DPG concentrations had lowered to 88% (20 degrees C) and 54% (28 degrees C) of initial levels, respectively. The difference was significant and was maintained for 28 days, although, at low levels from day 7 (28 degrees C) and day 14 (20 degrees C) of storage. ATP was maintained at the initial level in both groups during the first 6 h of storage but after component separation the levels were significantly higher in the 28 degrees C group during the first 5 days. The release of myeloperoxidase (MPO) was significantly higher in the non-cooled group than in the cooled group. Pre-separation holding for 6 h of whole blood at temperatures of 28 degrees C causes a great and rapid loss of 2,3-DPG and considerable formation of acid metabolites resulting in clearly subnormal 2,3-DPG levels even on day 1. Active pre-separation cooling to 20 degrees C is to be recommended.

  4. Identifying biomarkers for asthma diagnosis using targeted metabolomics approaches.

    PubMed

    Checkley, William; Deza, Maria P; Klawitter, Jost; Romero, Karina M; Klawitter, Jelena; Pollard, Suzanne L; Wise, Robert A; Christians, Uwe; Hansel, Nadia N

    2016-12-01

    The diagnosis of asthma in children is challenging and relies on a combination of clinical factors and biomarkers including methacholine challenge, lung function, bronchodilator responsiveness, and presence of airway inflammation. No single test is diagnostic. We sought to identify a pattern of inflammatory biomarkers that was unique to asthma using a targeted metabolomics approach combined with data science methods. We conducted a nested case-control study of 100 children living in a peri-urban community in Lima, Peru. We defined cases as children with current asthma, and controls as children with no prior history of asthma and normal lung function. We further categorized enrollment following a factorial design to enroll equal numbers of children as either overweight or not. We obtained a fasting venous blood sample to characterize a comprehensive panel of targeted markers using a metabolomics approach based on high performance liquid chromatography-mass spectrometry. A statistical comparison of targeted metabolites between children with asthma (n = 50) and healthy controls (n = 49) revealed distinct patterns in relative concentrations of several metabolites: children with asthma had approximately 40-50% lower relative concentrations of ascorbic acid, 2-isopropylmalic acid, shikimate-3-phosphate, and 6-phospho-d-gluconate when compared to children without asthma, and 70% lower relative concentrations of reduced glutathione (all p < 0.001 after Bonferroni correction). Moreover, a combination of 2-isopropylmalic acid and betaine strongly discriminated between children with asthma (2-isopropylmalic acid ≤ 13 077 normalized counts/second) and controls (2-isopropylmalic acid > 13 077 normalized counts/second and betaine ≤ 16 47 121 normalized counts/second). By using a metabolomics approach applied to serum, we were able to discriminate between children with and without asthma by revealing different metabolic patterns. These results suggest that

  5. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

    PubMed

    Cheng, Feixiong; Murray, James L; Zhao, Junfei; Sheng, Jinsong; Zhao, Zhongming; Rubin, Donald H

    2016-09-01

    Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

  6. Identifying mechanism-of-action targets for drugs and probes

    PubMed Central

    Gregori-Puigjané, Elisabet; Setola, Vincent; Hert, Jérôme; Crews, Brenda A.; Irwin, John J.; Lounkine, Eugen; Marnett, Lawrence; Roth, Bryan L.; Shoichet, Brian K.

    2012-01-01

    Notwithstanding their key roles in therapy and as biological probes, 7% of approved drugs are purported to have no known primary target, and up to 18% lack a well-defined mechanism of action. Using a chemoinformatics approach, we sought to “de-orphanize” drugs that lack primary targets. Surprisingly, targets could be easily predicted for many: Whereas these targets were not known to us nor to the common databases, most could be confirmed by literature search, leaving only 13 Food and Drug Administration—approved drugs with unknown targets; the number of drugs without molecular targets likely is far fewer than reported. The number of worldwide drugs without reasonable molecular targets similarly dropped, from 352 (25%) to 44 (4%). Nevertheless, there remained at least seven drugs for which reasonable mechanism-of-action targets were unknown but could be predicted, including the antitussives clemastine, cloperastine, and nepinalone; the antiemetic benzquinamide; the muscle relaxant cyclobenzaprine; the analgesic nefopam; and the immunomodulator lobenzarit. For each, predicted targets were confirmed experimentally, with affinities within their physiological concentration ranges. Turning this question on its head, we next asked which drugs were specific enough to act as chemical probes. Over 100 drugs met the standard criteria for probes, and 40 did so by more stringent criteria. A chemical information approach to drug-target association can guide therapeutic development and reveal applications to probe biology, a focus of much current interest. PMID:22711801

  7. Cutting needle biopsy combined with immunohistochemical study of myeloperoxidase for the diagnosis of histiocytic necrotizing lymphadenitis.

    PubMed

    Hanakawa, Hiroyuki; Orita, Yorihisa; Sato, Yasuharu; Takeuchi, Mai; Ohno, Kyotaro; Iwaki, Noriko; Ito, Toshihiro; Nishizaki, Kazunori; Yoshino, Tadashi

    2013-12-01

    Cutting needle biopsy (CNB) combined with immunohistochemical study of myeloperoxidase (MPO) is a useful minimally invasive diagnostic procedure for histiocytic necrotizing lymphadenitis (HNL). HNL is mainly diagnosed by pathological findings of open surgical biopsy (OSB) specimens. Recently the appearance of anti-MPO positive histiocytes has been reported as a highly specific pathological diagnosis for HNL. Considering the cosmetic impact and burden on the patients, we performed CNB combined with immunohistochemical study of MPO for the diagnosis of HNL. Few studies have reported the utility of this method in the diagnosis of HNL. A retrospective study was conducted using clinical data from 20 HNL patients. CNB was performed in 8 patients and OSB in 13 (OSB after CNB in 1). MPO-positive histiocytes were observed in all of the 20 cases. The accuracy of the diagnoses was finally confirmed by the clinical courses in all cases.

  8. Five-Year Longitudinal Assessment (2008 to 2012) of E-101 Solution Activity against Clinical Target and Antimicrobial-Resistant Pathogens

    PubMed Central

    Pillar, Chris M.; Sahm, Daniel F.; O'Hanley, Peter; Stephens, Jackson T.

    2014-01-01

    This study summarizes the topical E-101 solution susceptibility testing results for 760 Gram-positive and Gram-negative target pathogens collected from 75 U.S. sites between 2008 and 2012 and 103 ESKAPE pathogens. E-101 solution maintained potent activity against all bacterial species studied for each year tested, with MICs ranging from <0.008 to 0.25 μg porcine myeloperoxidase (pMPO)/ml. These results confirm that E-101 solution retains its potent broad-spectrum activity against U.S. clinical isolates and organisms with challenging resistance phenotypes. PMID:24841272

  9. Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

    PubMed Central

    Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

    2016-01-01

    Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central

  10. Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

    PubMed

    Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

    2016-11-01

    Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

  11. PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3′ UTRs and coding sequences

    PubMed Central

    Šulc, Miroslav; Marín, Ray M.; Robins, Harlan S.; Vaníček, Jiří

    2015-01-01

    The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3′ untranslated regions (3′ UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3′ UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA–mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA–mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. PMID:25948580

  12. Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

    PubMed

    Crisan, D; Anstett, M J

    1995-07-01

    Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

  13. Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo*

    PubMed Central

    Crouch, Erika C.; Hirche, Tim O.; Shao, Baohai; Boxio, Rachel; Wartelle, Julien; Benabid, Rym; McDonald, Barbara; Heinecke, Jay; Matalon, Sadis; Belaaouaj, Azzaq

    2010-01-01

    Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H2O2-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity. PMID:20228064

  14. Predicting essential genes for identifying potential drug targets in Aspergillus fumigatus.

    PubMed

    Lu, Yao; Deng, Jingyuan; Rhodes, Judith C; Lu, Hui; Lu, Long Jason

    2014-06-01

    Aspergillus fumigatus (Af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. Despite current therapeutic options, mortality associated with invasive Af infections remains unacceptably high, increasing 357% since 1980. Therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. Thus, as noted in a recent review, "the identification of essential genes in fungi represents a crucial step in the development of new antifungal drugs". Expanding the target space by rapidly identifying new essential genes has thus been described as "the most important task of genomics-based target validation". In previous research, we were the first to show that essential gene annotation can be reliably transferred between distantly related four Prokaryotic species. In this study, we extend our machine learning approach to the much more complex Eukaryotic fungal species. A compendium of essential genes is predicted in Af by transferring known essential gene annotations from another filamentous fungus Neurospora crassa. This approach predicts essential genes by integrating diverse types of intrinsic and context-dependent genomic features encoded in microbial genomes. The predicted essential datasets contained 1674 genes. We validated our results by comparing our predictions with known essential genes in Af, comparing our predictions with those predicted by homology mapping, and conducting conditional expressed alleles. We applied several layers of filters and selected a set of potential drug targets from the predicted essential genes. Finally, we have conducted wet lab knockout experiments to verify our predictions, which further validates the accuracy and wide applicability of the machine learning approach. The approach presented here significantly extended our ability to predict essential genes beyond orthologs and made it possible to

  15. Identifying Minefields and Verifying Clearance: Adapting Statistical Methods for UXO Target Detection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gilbert, Richard O.; O'Brien, Robert F.; Wilson, John E.

    2003-09-01

    It may not be feasible to completely survey large tracts of land suspected of containing minefields. It is desirable to develop a characterization protocol that will confidently identify minefields within these large land tracts if they exist. Naturally, surveying areas of greatest concern and most likely locations would be necessary but will not provide the needed confidence that an unknown minefield had not eluded detection. Once minefields are detected, methods are needed to bound the area that will require detailed mine detection surveys. The US Department of Defense Strategic Environmental Research and Development Program (SERDP) is sponsoring the development ofmore » statistical survey methods and tools for detecting potential UXO targets. These methods may be directly applicable to demining efforts. Statistical methods are employed to determine the optimal geophysical survey transect spacing to have confidence of detecting target areas of a critical size, shape, and anomaly density. Other methods under development determine the proportion of a land area that must be surveyed to confidently conclude that there are no UXO present. Adaptive sampling schemes are also being developed as an approach for bounding the target areas. These methods and tools will be presented and the status of relevant research in this area will be discussed.« less

  16. Association between Myeloperoxidase Levels and Risk of Insulin Resistance in Egyptian Obese Women

    PubMed Central

    Zaki, Moushira; Basha, Walaa; Reyad, Hanaa; Mohamed, Ramy; Hassan, Naglaa; Kholousi, Shams

    2018-01-01

    BACKGROUND: Myeloperoxidase (MPO) is an enzyme involved in the pathogenesis of several diseases. AIM: The current study aimed to investigate serum MPO levels in obese Egyptian women and assess its relation with insulin resistance (IR) and other biochemical risk parameters. METHODS: The study included 80 obese women and 50 age-and-sex-matched healthy controls. Insulin resistance (IR) was evaluated by the Homeostasis Model Assessment-Insulin Resistance (HOMA-IR). Serum MPO, fasting glucose, insulin and blood lipids and anthropometry were measured. Obese cases were divided into three groups based on MPO tertiles. ROC analysis was performed to obtain the optimal cut-off values of MPO to predicate IR in obese women. RESULTS: The mean serum MPO was significantly higher in obese cases than controls. Cases in the highest MPO tertile had higher HOMA-IR, blood lipids and pressure levels compared with those in the lower tertile. The cutoff point of MPO was > 87.8 (ng/mL) and area under curves was 0.82 (p < 0.01) for diagnosis of IR. MPO levels were higher in obese Egyptian women than healthy controls. CONCLUSION: Elevation of MPO was associated with abnormal metabolic parameters. MPO might be used as an earlier biomarker for IR and metabolic disturbance in obese women. PMID:29731928

  17. Myeloperoxidase-derived 2-chlorofatty acids contribute to human sepsis mortality via acute respiratory distress syndrome.

    PubMed

    Meyer, Nuala J; Reilly, John P; Feng, Rui; Christie, Jason D; Hazen, Stanley L; Albert, Carolyn J; Franke, Jacob D; Hartman, Celine L; McHowat, Jane; Ford, David A

    2017-12-07

    Sepsis-associated acute respiratory distress syndrome (ARDS) is characterized by neutrophilic inflammation and poor survival. Since neutrophil myeloperoxidase (MPO) activity leads to increased plasma 2-chlorofatty acid (2-ClFA) levels, we hypothesized that plasma concentrations of 2-ClFAs would associate with ARDS and mortality in subjects with sepsis. In sequential consenting patients with sepsis, free 2-ClFA levels were significantly associated with ARDS, and with 30-day mortality, for each log increase in free 2-chlorostearic acid. Plasma MPO was not associated with either ARDS or 30-day mortality but was correlated with 2-ClFA levels. Addition of plasma 2-ClFA levels to the APACHE III score improved prediction for ARDS. Plasma 2-ClFA levels correlated with plasma levels of angiopoietin-2, E selectin, and soluble thrombomodulin. Endothelial cells treated with 2-ClFA responded with increased adhesion molecule surface expression, increased angiopoietin-2 release, and dose-dependent endothelial permeability. Our results suggest that 2-ClFAs derived from neutrophil MPO-catalyzed oxidation contribute to pulmonary endothelial injury and have prognostic utility in sepsis-associated ARDS.

  18. Systems pharmacology identifies drug targets for Stargardt disease–associated retinal degeneration

    PubMed Central

    Chen, Yu; Palczewska, Grazyna; Mustafi, Debarshi; Golczak, Marcin; Dong, Zhiqian; Sawada, Osamu; Maeda, Tadao; Maeda, Akiko; Palczewski, Krzysztof

    2013-01-01

    A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases. PMID:24231350

  19. Identify mutation in amyotrophic lateral sclerosis cases using HaloPlex target enrichment system.

    PubMed

    Liu, Zhi-Jun; Li, Hong-Fu; Tan, Guo-He; Tao, Qing-Qing; Ni, Wang; Cheng, Xue-Wen; Xiong, Zhi-Qi; Wu, Zhi-Ying

    2014-12-01

    To date, at least 18 causative genes have been identified in amyotrophic lateral sclerosis (ALS). Because of the clinical and genetic heterogeneity, molecular diagnosis for ALS faces great challenges. HaloPlex target enrichment system is a new targeted sequencing approach, which can detect already known mutations or candidate genes. We performed this approach to screen 18 causative genes of ALS, including SOD1, SETX, FUS, ANG, TARDBP, ALS2, FIG4, VAPB, OPTN, DAO, VCP, UBQLN2, SPG11, SIGMAR1, DCTN1, SQSTM1, PFN1, and CHMP2B in 8 ALS probands. Using this approach, we got an average of 9.5 synonymous or missense mutations per sample. After validation by Sanger sequencing, we identified 3 documented SOD1 mutations (p.F21C, p.G148D, and p.C147R) and 1 novel DCTN1 p.G59R mutation in 4 probands. The novel DCTN1 mutation appeared to segregate with the disease in the pedigree and was absent in 200 control subjects. The high throughput and efficiency of this approach indicated that it could be applied to diagnose ALS and other inherited diseases with multiple causative genes in clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells.

    PubMed

    Somanath, Priyanka; Herndon Klein, Rachel; Knoepfler, Paul S

    2017-01-01

    The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

  1. Identifying On-Orbit Test Targets for Space Fence Operational Testing

    NASA Astrophysics Data System (ADS)

    Pechkis, D.; Pacheco, N.; Botting, T.

    2014-09-01

    Space Fence will be an integrated system of two ground-based, S-band (2 to 4 GHz) phased-array radars located in Kwajalein and perhaps Western Australia [1]. Space Fence will cooperate with other Space Surveillance Network sensors to provide space object tracking and radar characterization data to support U.S. Strategic Command space object catalog maintenance and other space situational awareness needs. We present a rigorous statistical test design intended to test Space Fence to the letter of the program requirements as well as to characterize the system performance across the entire operational envelope. The design uses altitude, size, and inclination as independent factors in statistical tests of dependent variables (e.g., observation accuracy) linked to requirements. The analysis derives the type and number of necessary test targets. Comparing the resulting sample sizes with the number of currently known targets, we identify those areas where modelling and simulation methods are needed. Assuming hypothetical Kwajalein radar coverage and a conservative number of radar passes per object per day, we conclude that tests involving real-world space objects should take no more than 25 days to evaluate all operational requirements; almost 60 percent of the requirements can be tested in a single day and nearly 90 percent can be tested in one week or less. Reference: [1] L. Haines and P. Phu, Space Fence PDR Concept Development Phase, 2011 AMOS Conference Technical Papers.

  2. High-throughput screening identifies microRNAs that target Nox2 and improve function after acute myocardial infarction.

    PubMed

    Yang, Junyu; Brown, Milton E; Zhang, Hanshuo; Martinez, Mario; Zhao, Zhihua; Bhutani, Srishti; Yin, Shenyi; Trac, David; Xi, Jianzhong Jeff; Davis, Michael E

    2017-05-01

    Myocardial infarction (MI) is the most common cause of heart failure. Excessive production of ROS plays a key role in the pathogenesis of cardiac remodeling after MI. NADPH with NADPH oxidase (Nox)2 as the catalytic subunit is a major source of superoxide production, and expression is significantly increased in the infarcted myocardium, especially by infiltrating macrophages. While microRNAs (miRNAs) are potent regulators of gene expression and play an important role in heart disease, there still lacks efficient ways to identify miRNAs that target important pathological genes for treating MI. Thus, the overall objective was to establish a miRNA screening and delivery system for improving heart function after MI using Nox2 as a critical target. With the use of the miRNA-target screening system composed of a self-assembled cell microarray (SAMcell), three miRNAs, miR-106b, miR-148b, and miR-204, were identified that could regulate Nox2 expression and its downstream products in both human and mouse macrophages. Each of these miRNAs were encapsulated into polyketal (PK3) nanoparticles that could effectively deliver miRNAs into macrophages. Both in vitro and in vivo studies in mice confirmed that PK3-miRNAs particles could inhibit Nox2 expression and activity and significantly improve infarct size and acute cardiac function after MI. In conclusion, our results show that miR-106b, miR-148b, and miR-204 were able to improve heart function after myocardial infarction in mice by targeting Nox2 and possibly altering inflammatory cytokine production. This screening system and delivery method could have broader implications for miRNA-mediated therapeutics for cardiovascular and other diseases. NEW & NOTEWORTHY NADPH oxidase (Nox)2 is a promising target for treating cardiovascular disease, but there are no specific inhibitors. Finding endogenous signals that can target Nox2 and other inflammatory molecules is of great interest. In this study, we used high-throughput screening

  3. ESR and X-ray Structure Investigations on the Binding and Mechanism of Inhibition of the Native State of Myeloperoxidase with Low Molecular Weight Fragments

    DOE PAGES

    Chavali, Balagopalakrishna; Masquelin, Thierry; Nilges, Mark J.; ...

    2015-05-19

    As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase (hMPO) offers an attractive protein target to modulate the inflammation of the host tissue through suitable inhibitors. We describe a novel methodology of using low temperature ESR spectroscopy (6 K) and FAST™ technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO. Also, our initial efforts to profile molecules on the inhibition of MPO-initiated nitration of the Apo-A1 peptide (AEYHAKATEHL) assay showed several potent (with sub-micro molar IC50s) but spurious inhibitors that either do not bindmore » to the heme pocket in the enzyme or retain high (>50 %) anti oxidant potential. Such molecules when taken forward for X-ray did not yield inhibitor-bound co-crystals. We then used ESR to confirm direct binding to the native state enzyme, by measuring the binding-induced shift in the electronic parameter g to rank order the molecules. Molecules with a higher rank order—those with g-shift R relative ≥15—yielded well-formed protein-bound crystals (n = 33 structures). The co-crystal structure with the LSN217331 inhibitor reveals that the chlorophenyl group projects away from the heme along the edges of the Phe366 and Phe407 side chain phenyl rings thereby sterically restricting the access to the heme by the substrates like H 2O 2. Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). In conclusion, our results point to a viable path forward to target the native state of MPO to tame local inflammation.« less

  4. Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.

    PubMed

    Ng, Samuel Y; Yoshida, Noriaki; Christie, Amanda L; Ghandi, Mahmoud; Dharia, Neekesh V; Dempster, Joshua; Murakami, Mark; Shigemori, Kay; Morrow, Sara N; Van Scoyk, Alexandria; Cordero, Nicolas A; Stevenson, Kristen E; Puligandla, Maneka; Haas, Brian; Lo, Christopher; Meyers, Robin; Gao, Galen; Cherniack, Andrew; Louissaint, Abner; Nardi, Valentina; Thorner, Aaron R; Long, Henry; Qiu, Xintao; Morgan, Elizabeth A; Dorfman, David M; Fiore, Danilo; Jang, Julie; Epstein, Alan L; Dogan, Ahmet; Zhang, Yanming; Horwitz, Steven M; Jacobsen, Eric D; Santiago, Solimar; Ren, Jian-Guo; Guerlavais, Vincent; Annis, D Allen; Aivado, Manuel; Saleh, Mansoor N; Mehta, Amitkumar; Tsherniak, Aviad; Root, David; Vazquez, Francisca; Hahn, William C; Inghirami, Giorgio; Aster, Jon C; Weinstock, David M; Koch, Raphael

    2018-05-22

    T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.

  5. Acoustic Parametric Array for Identifying Standoff Targets

    NASA Astrophysics Data System (ADS)

    Hinders, M. K.; Rudd, K. E.

    2010-02-01

    An integrated simulation method for investigating nonlinear sound beams and 3D acoustic scattering from any combination of complicated objects is presented. A standard finite-difference simulation method is used to model pulsed nonlinear sound propagation from a source to a scattering target via the KZK equation. Then, a parallel 3D acoustic simulation method based on the finite integration technique is used to model the acoustic wave interaction with the target. Any combination of objects and material layers can be placed into the 3D simulation space to study the resulting interaction. Several example simulations are presented to demonstrate the simulation method and 3D visualization techniques. The combined simulation method is validated by comparing experimental and simulation data and a demonstration of how this combined simulation method assisted in the development of a nonlinear acoustic concealed weapons detector is also presented.

  6. Use of eQTL Analysis for the Discovery of Target Genes Identified by GWAS

    DTIC Science & Technology

    2013-04-01

    the biologic pathways affected by these inherited factors, and ultimately to identify targets for disease prediction, risk stratification and...quality using an Agilent chip technology. Cases having a RIN number of 7.0 or greater were considered good quality. Once completed, the optimum set of...AD_________________ Award Number: W81XWH-11-1-0261 TITLE: Use of eQTL Analysis for the Discovery of

  7. TP53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing.

    PubMed

    Zheng, Huili; Wang, Yan; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Zhang, Guangchun; Cao, Weihai; Li, Jingwen; Liu, Lifeng; Liu, Zhencong; Zhang, Chao; Lou, Feng; Liu, Zhiyuan; Li, Yangyang; Shi, Zhenfen; Zhang, Jingbo; Zhang, Dandan; Sun, Hong; Dong, Haichao; Dong, Zhishou; Guo, Baishuai; Yan, H E; Lu, Qingyu; Huang, Xue; Chen, Si-Yi

    2016-01-01

    Esophageal cancer (EC) is a common malignancy with significant morbidity and mortality. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in EC that may serve as biomarkers might help predict patient outcome and guide treatment. Traditionally, personalized cancer DNA sequencing was impractical and expensive. Recent technological advancements have made targeted DNA sequencing more cost- and time-effective with reliable results. This technology may be useful for clinicians to direct patient treatment. The Ion PGM and AmpliSeq Cancer Panel was used to identify mutations at 737 hotspot loci of 45 cancer-related genes in 64 EC samples from Chinese patients. Frequent mutations were found in TP53 and less frequent mutations in PIK3CA, FBXW7 and KRAS. These results demonstrate that targeted sequencing can reliably identify mutations in individual tumors that make this technology a possibility for clinical use. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  8. A Behavioral Intervention to Reduce Child Exposure to Indoor Air Pollution: Identifying Possible Target Behaviors

    ERIC Educational Resources Information Center

    Barnes, Brendon R.; Mathee, Angela; Shafritz, Lonna B.; Krieger, Laurie; Zimicki, Susan

    2004-01-01

    Indoor air pollution has been causally linked to acute lower respiratory infections in children younger than 5. The aim of this study was to identify target behaviors for a behavioral intervention to reduce child exposure to indoor air pollution by attempting to answer two research questions: Which behaviors are protective of child respiratory…

  9. Kidney disease models: tools to identify mechanisms and potential therapeutic targets

    PubMed Central

    Bao, Yin-Wu; Yuan, Yuan; Chen, Jiang-Hua; Lin, Wei-Qiang

    2018-01-01

    Acute kidney injury (AKI) and chronic kidney disease (CKD) are worldwide public health problems affecting millions of people and have rapidly increased in prevalence in recent years. Due to the multiple causes of renal failure, many animal models have been developed to advance our understanding of human nephropathy. Among these experimental models, rodents have been extensively used to enable mechanistic understanding of kidney disease induction and progression, as well as to identify potential targets for therapy. In this review, we discuss AKI models induced by surgical operation and drugs or toxins, as well as a variety of CKD models (mainly genetically modified mouse models). Results from recent and ongoing clinical trials and conceptual advances derived from animal models are also explored. PMID:29515089

  10. Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) Identifies Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Targets in Endothelial Cells.

    PubMed

    Gay, Lauren A; Sethuraman, Sunantha; Thomas, Merin; Turner, Peter C; Renne, Rolf

    2018-04-15

    Kaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3' untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis. IMPORTANCE KS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will

  11. Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy.

    PubMed

    Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo; Xu, Ge-Zhi

    2017-01-01

    Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. We identified two novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.

  12. Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

    PubMed Central

    Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

    2017-01-01

    Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

  13. Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

    PubMed

    Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

    2003-10-15

    SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis

  14. A new method of identifying target groups for pronatalist policy applied to Australia.

    PubMed

    Chen, Mengni; Lloyd, Chris J; Yip, Paul S F

    2018-01-01

    A country's total fertility rate (TFR) depends on many factors. Attributing changes in TFR to changes of policy is difficult, as they could easily be correlated with changes in the unmeasured drivers of TFR. A case in point is Australia where both pronatalist effort and TFR increased in lock step from 2001 to 2008 and then decreased. The global financial crisis or other unobserved confounders might explain both the reducing TFR and pronatalist incentives after 2008. Therefore, it is difficult to estimate causal effects of policy using econometric techniques. The aim of this study is to instead look at the structure of the population to identify which subgroups most influence TFR. Specifically, we build a stochastic model relating TFR to the fertility rates of various subgroups and calculate elasticity of TFR with respect to each rate. For each subgroup, the ratio of its elasticity to its group size is used to evaluate the subgroup's potential cost effectiveness as a pronatalist target. In addition, we measure the historical stability of group fertility rates, which measures propensity to change. Groups with a high effectiveness ratio and also high propensity to change are natural policy targets. We applied this new method to Australian data on fertility rates broken down by parity, age and marital status. The results show that targeting parity 3+ is more cost-effective than lower parities. This study contributes to the literature on pronatalist policies by investigating the targeting of policies, and generates important implications for formulating cost-effective policies.

  15. A new method of identifying target groups for pronatalist policy applied to Australia

    PubMed Central

    Chen, Mengni; Lloyd, Chris J.

    2018-01-01

    A country’s total fertility rate (TFR) depends on many factors. Attributing changes in TFR to changes of policy is difficult, as they could easily be correlated with changes in the unmeasured drivers of TFR. A case in point is Australia where both pronatalist effort and TFR increased in lock step from 2001 to 2008 and then decreased. The global financial crisis or other unobserved confounders might explain both the reducing TFR and pronatalist incentives after 2008. Therefore, it is difficult to estimate causal effects of policy using econometric techniques. The aim of this study is to instead look at the structure of the population to identify which subgroups most influence TFR. Specifically, we build a stochastic model relating TFR to the fertility rates of various subgroups and calculate elasticity of TFR with respect to each rate. For each subgroup, the ratio of its elasticity to its group size is used to evaluate the subgroup’s potential cost effectiveness as a pronatalist target. In addition, we measure the historical stability of group fertility rates, which measures propensity to change. Groups with a high effectiveness ratio and also high propensity to change are natural policy targets. We applied this new method to Australian data on fertility rates broken down by parity, age and marital status. The results show that targeting parity 3+ is more cost-effective than lower parities. This study contributes to the literature on pronatalist policies by investigating the targeting of policies, and generates important implications for formulating cost-effective policies. PMID:29425220

  16. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    NASA Astrophysics Data System (ADS)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  17. Neutrophil collagenase, gelatinase, and myeloperoxidase in tears of patients with stevens-johnson syndrome and ocular cicatricial pemphigoid.

    PubMed

    Arafat, Samer N; Suelves, Ana M; Spurr-Michaud, Sandra; Chodosh, James; Foster, C Stephen; Dohlman, Claes H; Gipson, Ilene K

    2014-01-01

    To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP). Prospective, noninterventional cohort study. Four SJS patients (7 eyes), 19 OCP patients (37 eyes), and 20 healthy controls who underwent phacoemulsification (40 eyes). Tear washes were collected from all patients and were analyzed for levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 using multianalyte bead-based enzyme-linked immunosorbent assays. Total MMP activity was determined using a fluorometric assay. Correlation studies were performed between the various analytes within study groups. Levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 (in nanograms per microgram of protein) and total MMP activity (in relative fluorescent units per minute per microgram of protein) in tears; MMP-8-to-TIMP-1 ratio; MMP-9-to-TIMP-1 ratio; and the correlations between MMP-8 and MMP-9 and both MMP and MPO. MMP-8, MMP-9, and MPO levels were elevated significantly in SJS and OCP tears (SJS>OCP) when compared with controls. The MMP activity was highest in SJS patients, whereas OCP patients and controls showed lower and similar activities. The TIMP-1 levels were decreased in SJS and OCP patients when compared with those in controls, with levels in OCP patients reaching significance. The MMP-8-to-TIMP-1 and MMP-9-to-TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS>OCP) when compared with those of controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels, and MMP-8 correlated with MPO, but it did not reach significance in SJS patients. There was no relationship between MMP-7 and MPO. Because MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes, including MMP-9

  18. The Near-Earth Object Human Space Flight Accessible Targets Study (NHATS) List of Near-Earth Asteroids: Identifying Potential Targets for Future Exploration

    NASA Astrophysics Data System (ADS)

    Abell, Paul; Barbee, B. W.; Mink, R. G.; Adamo, D. R.; Alberding, C. M.; Mazanek, D. D.; Johnson, L. N.; Yeomans, D. K.; Chodas, P. W.; Chamberlin, A. B.; Benner, L. A. M.; Drake, B. G.; Friedensen, V. P.

    2012-10-01

    Introduction: Much attention has recently been focused on human exploration of near-Earth asteroids (NEAs). Detailed planning for deep space exploration and identification of potential NEA targets for human space flight requires selecting objects from the growing list of known NEAs. NASA therefore initiated the Near-Earth Object Human Space Flight Accessible Target Study (NHATS), which uses dynamical trajectory performance constraints to identify potentially accessible NEAs. Accessibility Criteria: Future NASA human space flight capability is being defined while the Orion Multi-Purpose Crew Vehicle and Space Launch System are under development. Velocity change and mission duration are two of the most critical factors in any human spaceflight endeavor, so the most accessible NEAs tend to be those with orbits similar to Earth’s. To be classified as NHATS-compliant, a NEA must offer at least one round-trip trajectory solution satisfying purposely inclusive constraints, including total mission change in velocity ≤ 12 km/s, mission duration ≤ 450 days (with at least 8 days at the NEA), Earth departure between Jan 1, 2015 and Dec 31, 2040, Earth departure C3 ≤ 60 km2/s2, and Earth return atmospheric entry speed ≤ 12 km/s. Monitoring and Updates: The NHATS list of potentially accessible targets is continuously updated as NEAs are discovered and orbit solutions for known NEAs are improved. The current list of accessible NEAs identified as potentially viable for future human exploration under the NHATS criteria is available to the international community via a website maintained by NASA’s NEO Program Office (http://neo.jpl.nasa.gov/nhats/). This website also lists predicted optical and radar observing opportunities for each NHATS-compliant NEA to facilitate acquisition of follow-up observations. Conclusions: This list of NEAs will be useful for analyzing robotic mission opportunities, identifying optimal round trip human space flight trajectories, and

  19. In-situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

    PubMed

    Liang, Huei-Chen; Liu, Yi-Chen; Chen, Hsin; Ku, Ming Chun; Do, Quynh-Trang; Wang, Chih-Yen; Tzeng, Shun-Fen; Chen, Shu-Hui

    2018-06-13

    Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in-situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified (n  2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 non-specific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9) which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

  20. Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

    PubMed Central

    2014-01-01

    Background Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. Methods To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. Results Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. Conclusions Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma. PMID:24661910

  1. A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection in the Drosophila neuromuscular system

    PubMed Central

    Kurusu, Mitsuhiko; Cording, Amy; Taniguchi, Misako; Menon, Kaushiki; Suzuki, Emiko; Zinn, Kai

    2008-01-01

    Summary In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron. PMID:18817735

  2. Identifying mechanisms for facilitating knowledge to action strategies targeting the built environment.

    PubMed

    Fazli, Ghazal S; Creatore, Maria I; Matheson, Flora I; Guilcher, Sara; Kaufman-Shriqui, Vered; Manson, Heather; Johns, Ashley; Booth, Gillian L

    2017-01-03

    In recent years, obesity-related diseases have been on the rise globally resulting in major challenges for health systems and society as a whole. Emerging research in population health suggests that interventions targeting the built environment may help reduce the burden of obesity and type 2 diabetes. However, translation of the evidence on the built environment into effective policy and planning changes requires engagement and collaboration between multiple sectors and government agencies for designing neighborhoods that are more conducive to healthy and active living. In this study, we identified knowledge gaps and other barriers to evidence-based decision-making and policy development related to the built environment; as well as the infrastructure, processes, and mechanisms needed to drive policy changes in this area. We conducted a qualitative thematic analysis of data collected through consultations with a broad group of stakeholders (N = 42) from Southern Ontario, Canada, within various sectors (public health, urban planning, and transportation) and levels of government (federal, provincial, and municipalities). Relevant themes were classified based on the specific phase of the knowledge-to-action cycle (research, translation, and implementation) in which they were most closely aligned. We identified 5 themes including: 1) the need for policy-informed and actionable research (e.g. health economic analyses and policy evaluations); 2) impactful messaging that targets all relevant sectors to create the political will necessary to drive policy change; 3) common measures and tools to increase capacity for monitoring and surveillance of built environment changes; (4) intersectoral collaboration and alignment within and between levels of government to enable collective actions and provide mechanisms for sharing of resources and expertise, (5) aligning public and private sector priorities to generate public demand and support for community action; and, (6

  3. Functional precision medicine identifies novel druggable targets and therapeutic options in head and neck cancer. | Office of Cancer Genomics

    Cancer.gov

    Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low passage tumor cells derived from a patient with a treatmentresistant HPV-negative HNSCC.

  4. Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

    PubMed Central

    Coronnello, Claudia; Hartmaier, Ryan; Arora, Arshi; Huleihel, Luai; Pandit, Kusum V.; Bais, Abha S.; Butterworth, Michael; Kaminski, Naftali; Stormo, Gary D.; Oesterreich, Steffi; Benos, Panayiotis V.

    2012-01-01

    MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for mi

  5. COMPARATIVE DIVERSITY OF FECAL BACTERIA IN AGRICULTURALLY SIGNIFICANT ANIMALS TO IDENTIFY ALTERNATIVE TARGETS FOR MICROBIAL SOURCE TRACKING

    EPA Science Inventory

    Animals of agricultural significance contribute a large percentage of fecal pollution to waterways via runoff contamination. The premise of microbial source tracking is to utilize fecal bacteria to identify target populations which are directly correlated to specific animal feces...

  6. iTAR: a web server for identifying target genes of transcription factors using ChIP-seq or ChIP-chip data.

    PubMed

    Yang, Chia-Chun; Andrews, Erik H; Chen, Min-Hsuan; Wang, Wan-Yu; Chen, Jeremy J W; Gerstein, Mark; Liu, Chun-Chi; Cheng, Chao

    2016-08-12

    Chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) or microarray hybridization (ChIP-chip) has been widely used to determine the genomic occupation of transcription factors (TFs). We have previously developed a probabilistic method, called TIP (Target Identification from Profiles), to identify TF target genes using ChIP-seq/ChIP-chip data. To achieve high specificity, TIP applies a conservative method to estimate significance of target genes, with the trade-off being a relatively low sensitivity of target gene identification compared to other methods. Additionally, TIP's output does not render binding-peak locations or intensity, information highly useful for visualization and general experimental biological use, while the variability of ChIP-seq/ChIP-chip file formats has made input into TIP more difficult than desired. To improve upon these facets, here we present are fined TIP with key extensions. First, it implements a Gaussian mixture model for p-value estimation, increasing target gene identification sensitivity and more accurately capturing the shape of TF binding profile distributions. Second, it enables the incorporation of TF binding-peak data by identifying their locations in significant target gene promoter regions and quantifies their strengths. Finally, for full ease of implementation we have incorporated it into a web server ( http://syslab3.nchu.edu.tw/iTAR/ ) that enables flexibility of input file format, can be used across multiple species and genome assembly versions, and is freely available for public use. The web server additionally performs GO enrichment analysis for the identified target genes to reveal the potential function of the corresponding TF. The iTAR web server provides a user-friendly interface and supports target gene identification in seven species, ranging from yeast to human. To facilitate investigating the quality of ChIP-seq/ChIP-chip data, the web server generates the chart of the

  7. Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

    PubMed Central

    Peri, Suraj; Devarajan, Karthik; Yang, Dong-Hua; Knudson, Alfred G.; Balachandran, Siddharth

    2013-01-01

    Objective To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes. Methods Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC. Results A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis. Conclusions These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently

  8. Systemic analysis of genome-wide expression profiles identified potential therapeutic targets of demethylation drugs for glioblastoma.

    PubMed

    Ning, Tongbo; Cui, Hao; Sun, Feng; Zou, Jidian

    2017-09-05

    Glioblastoma represents one of the most aggressive malignant brain tumors with high morbidity and motility. Demethylation drugs have been developed for its treatment with little efficacy has been observed. The purpose of this study was to screen therapeutic targets of demethylation drugs or bioactive molecules for glioblastoma through systemic bioinformatics analysis. We firstly downloaded genome-wide expression profiles from the Gene Expression Omnibus (GEO) and conducted the primary analysis through R software, mainly including preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differential expression genes (DEGs). Secondly, functional enrichment analysis was conducted via the Database for Annotation, Visualization and Integrated Discovery (DAVID) to explore biological processes involved in the development of glioblastoma. Thirdly, we constructed protein-protein interaction (PPI) network of interested genes and conducted cross analysis for multi datasets to obtain potential therapeutic targets for glioblastoma. Finally, we further confirmed the therapeutic targets through real-time RT-PCR. As a result, biological processes that related to cancer development, amino metabolism, immune response and etc. were found to be significantly enriched in genes that differential expression in glioblastoma and regulated by 5'aza-dC. Besides, network and cross analysis identified ACAT2, UFC1 and CYB5R1 as novel therapeutic targets of demethylation drugs which also confirmed by real time RT-PCR. In conclusions, our study identified several biological processes and genes that involved in the development of glioblastoma and regulated by 5'aza-dC, which would be helpful for the treatment of glioblastoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous‐cell carcinoma

    PubMed Central

    Syed, Nazia; Barbhuiya, Mustafa A.; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K.; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T. S. Keshava; Kumar, M. Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh

    2015-01-01

    Esophageal squamous‐cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early‐stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non‐neoplastic Het‐1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry‐based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA‐based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation. PMID:25366905

  10. Classification accuracy of claims-based methods for identifying providers failing to meet performance targets.

    PubMed

    Hubbard, Rebecca A; Benjamin-Johnson, Rhondee; Onega, Tracy; Smith-Bindman, Rebecca; Zhu, Weiwei; Fenton, Joshua J

    2015-01-15

    Quality assessment is critical for healthcare reform, but data sources are lacking for measurement of many important healthcare outcomes. With over 49 million people covered by Medicare as of 2010, Medicare claims data offer a potentially valuable source that could be used in targeted health care quality improvement efforts. However, little is known about the operating characteristics of provider profiling methods using claims-based outcome measures that may estimate provider performance with error. Motivated by the example of screening mammography performance, we compared approaches to identifying providers failing to meet guideline targets using Medicare claims data. We used data from the Breast Cancer Surveillance Consortium and linked Medicare claims to compare claims-based and clinical estimates of cancer detection rate. We then demonstrated the performance of claim-based estimates across a broad range of operating characteristics using simulation studies. We found that identification of poor performing providers was extremely sensitive to algorithm specificity, with no approach identifying more than 65% of poor performing providers when claims-based measures had specificity of 0.995 or less. We conclude that claims have the potential to contribute important information on healthcare outcomes to quality improvement efforts. However, to achieve this potential, development of highly accurate claims-based outcome measures should remain a priority. Copyright © 2014 John Wiley & Sons, Ltd.

  11. Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

    PubMed

    Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

    2018-01-01

    Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date

  12. Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

    PubMed Central

    Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

    2018-01-01

    ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been

  13. Resveratrol Confers Protection against Rotenone-Induced Neurotoxicity by Modulating Myeloperoxidase Levels in Glial Cells

    PubMed Central

    Chang, Chi Young; Choi, Dong-Kug; Lee, Dae Kee; Hong, Young Jun; Park, Eun Jung

    2013-01-01

    Myeloperoxidase (MPO) functions as a key molecular component of the host defense system against diverse pathogens. We have previously reported that increased MPO levels and activity is a distinguishing feature of rotenone-exposed glial cells, and that either overactivation or deficiency of MPO leads to pathological conditions in the brain. Here, we provide that modulation of MPO levels in glia by resveratrol confers protective effects on rotenone-induced neurotoxicity. We show that resveratrol significantly reduced MPO levels but did not trigger abnormal nitric oxide (NO) production in microglia and astrocytes. Resveratrol-induced down-regulation of MPO, in the absence of an associated overproduction of NO, markedly attenuated rotenone-triggered inflammatory responses including phagocytic activity and reactive oxygen species production in primary microglia and astrocytes. In addition, impaired responses of primary mixed glia from Mpo −/− mice to rotenone were relieved by treatment with resveratrol. We further show that rotenone-induced neuronal injury, particularly dopaminergic cell death, was attenuated by resveratrol in neuron-glia co-cultures, but not in neurons cultured alone. Similar regulatory effects of resveratrol on MPO levels were observed in microglia treated with MPP+, another Parkinson’s disease-linked neurotoxin, supporting the beneficial effects of resveratrol on the brain. Collectively, our findings provide that resveratrol influences glial responses to rotenone by regulating both MPO and NO, and thus protects against rotenone-induced neuronal injury. PMID:23593274

  14. Researchers Use a Kinome Screen to Identify New Therapeutic Targets | Office of Cancer Genomics

    Cancer.gov

    The tumor suppressor p53 is mutated in over 50% of head and neck squamous cell carcinomas (HNSCC), yet there are currently no available therapies to target it. CTD2 researchers at the Fred Hutchison Cancer Research Center hypothesized that HNSCC cancer cells with p53 mutations are dependent on particular kinases for survival. In a study published in Clinical Cancer Research, they sought to identify these kinases using RNAi against known kinase genes in mouse and human cell lines.

  15. Myeloperoxidase in blood neutrophils during normal and abnormal menstrual cycles in women of reproductive age.

    PubMed

    Shibata, T; Sakamoto, J; Osaka, Y; Neyatani, N; Fujita, S; Oka, Y; Takagi, H; Mori, H; Fujita, H; Tanaka, Y; Sasagawa, T

    2017-04-01

    We previously reported that granulocyte colony-stimulating factor (G-CSF) plays a critical role in ovulation, suggesting that neutrophils may maintain ovulation. We assessed myeloperoxidase (MPO), a major and specific enzyme of neutrophils, in women with abnormal and normal menstrual cycles to clarify the relationship between MPO and ovulation. We analyzed MPO activity in blood neutrophils of women with abnormal menstrual cycles (indicative of anovulation, n = 12) and age- and body mass index-matched normal menstrual cycles (indicative of ovulation, n = 24) using two parameters as a marker of MPO, Neut X and mean peroxidase index (MPXI). MPO of women with abnormal menstrual cycles was significantly lower than that of women with normal menstrual cycles [Neut X: 62.6 ± 1.1 (mean ± standard error of the mean) vs. 66.2 ± 0.3, P = 0.009; MPXI: -0.54 ± 1.66 vs. 4.91 ± 0.53, P = 0.008]. Among women with normal menstrual cycles, MPO was highest in the follicular phase (Neut X: 67.0 ± 0.3; P = 0.033). The difference in MPO between women with abnormal and normal menstrual cycles and the upregulation of MPO before ovulation suggest that neutrophils and MPO are closely related to ovulation. © 2016 John Wiley & Sons Ltd.

  16. Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer.

    PubMed

    Chang, Hae Ryung; Nam, Seungyoon; Lee, Jinhyuk; Kim, Jin-Hee; Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

    2016-12-06

    Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer "Big Data" has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of "hit" compounds.

  17. Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord.

    PubMed

    Tamplin, Owen J; Cox, Brian J; Rossant, Janet

    2011-12-15

    The node and notochord are key tissues required for patterning of the vertebrate body plan. Understanding the gene regulatory network that drives their formation and function is therefore important. Foxa2 is a key transcription factor at the top of this genetic hierarchy and finding its targets will help us to better understand node and notochord development. We performed an extensive microarray-based gene expression screen using sorted embryonic notochord cells to identify early notochord-enriched genes. We validated their specificity to the node and notochord by whole mount in situ hybridization. This provides the largest available resource of notochord-expressed genes, and therefore candidate Foxa2 target genes in the notochord. Using existing Foxa2 ChIP-seq data from adult liver, we were able to identify a set of genes expressed in the notochord that had associated regions of Foxa2-bound chromatin. Given that Foxa2 is a pioneer transcription factor, we reasoned that these sites might represent notochord-specific enhancers. Candidate Foxa2-bound regions were tested for notochord specific enhancer function in a zebrafish reporter assay and 7 novel notochord enhancers were identified. Importantly, sequence conservation or predictive models could not have readily identified these regions. Mutation of putative Foxa2 binding elements in two of these novel enhancers abrogated reporter expression and confirmed their Foxa2 dependence. The combination of highly specific gene expression profiling and genome-wide ChIP analysis is a powerful means of understanding developmental pathways, even for small cell populations such as the notochord. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Using Market Research to Characterize College Students and Identify Potential Targets for Influencing Health Behaviors

    PubMed Central

    Berg, Carla J.; Ling, Pamela M.; Guo, Hongfei; Windle, Michael; Thomas, Janet L.; Ahluwalia, Jasjit S.; An, Lawrence C.

    2013-01-01

    Marketing campaigns, such as those developed by the tobacco industry, are based on market research, which defines segments of a population by assessing psychographic characteristics (i.e., attitudes, interests). This study uses a similar approach to define market segments of college smokers, to examine differences in their health behaviors (smoking, drinking, binge drinking, exercise, diet), and to determine the validity of these segments. A total of 2,265 undergraduate students aged 18–25 years completed a 108-item online survey in fall 2008 assessing demographic, psychographic (i.e., attitudes, interests), and health-related variables. Among the 753 students reporting past 30-day smoking, cluster analysis was conducted using 21 psychographic questions and identified three market segments – Stoic Individualists, Responsible Traditionalists, and Thrill-Seeking Socializers. We found that segment membership was related to frequency of alcohol use, binge drinking, and limiting dietary fat. We then developed three messages targeting each segment and conducted message testing to validate the segments on a subset of 73 smokers representing each segment in spring 2009. As hypothesized, each segment indicated greater relevance and salience for their respective message. These findings indicate that identifying qualitatively different subgroups of young adults through market research may inform the development of engaging interventions and health campaigns targeting college students. PMID:25264429

  19. Using Market Research to Characterize College Students and Identify Potential Targets for Influencing Health Behaviors.

    PubMed

    Berg, Carla J; Ling, Pamela M; Guo, Hongfei; Windle, Michael; Thomas, Janet L; Ahluwalia, Jasjit S; An, Lawrence C

    2010-12-01

    Marketing campaigns, such as those developed by the tobacco industry, are based on market research, which defines segments of a population by assessing psychographic characteristics (i.e., attitudes, interests). This study uses a similar approach to define market segments of college smokers, to examine differences in their health behaviors (smoking, drinking, binge drinking, exercise, diet), and to determine the validity of these segments. A total of 2,265 undergraduate students aged 18-25 years completed a 108-item online survey in fall 2008 assessing demographic, psychographic (i.e., attitudes, interests), and health-related variables. Among the 753 students reporting past 30-day smoking, cluster analysis was conducted using 21 psychographic questions and identified three market segments - Stoic Individualists, Responsible Traditionalists, and Thrill-Seeking Socializers. We found that segment membership was related to frequency of alcohol use, binge drinking, and limiting dietary fat. We then developed three messages targeting each segment and conducted message testing to validate the segments on a subset of 73 smokers representing each segment in spring 2009. As hypothesized, each segment indicated greater relevance and salience for their respective message. These findings indicate that identifying qualitatively different subgroups of young adults through market research may inform the development of engaging interventions and health campaigns targeting college students.

  20. 2D and 3D similarity landscape analysis identifies PARP as a novel off-target for the drug Vatalanib.

    PubMed

    Gohlke, Bjoern-Oliver; Overkamp, Tim; Richter, Anja; Richter, Antje; Daniel, Peter T; Gillissen, Bernd; Preissner, Robert

    2015-09-24

    Searching for two-dimensional (2D) structural similarities is a useful tool to identify new active compounds in drug-discovery programs. However, as 2D similarity measures neglect important structural and functional features, similarity by 2D might be underestimated. In the present study, we used combined 2D and three-dimensional (3D) similarity comparisons to reveal possible new functions and/or side-effects of known bioactive compounds. We utilised more than 10,000 compounds from the SuperTarget database with known inhibition values for twelve different anti-cancer targets. We performed all-against-all comparisons resulting in 2D similarity landscapes. Among the regions with low 2D similarity scores are inhibitors of vascular endothelial growth factor receptor (VEGFR) and inhibitors of poly ADP-ribose polymerase (PARP). To demonstrate that 3D landscape comparison can identify similarities, which are untraceable in 2D similarity comparisons, we analysed this region in more detail. This 3D analysis showed the unexpected structural similarity between inhibitors of VEGFR and inhibitors of PARP. Among the VEGFR inhibitors that show similarities to PARP inhibitors was Vatalanib, an oral "multi-targeted" small molecule protein kinase inhibitor being studied in phase-III clinical trials in cancer therapy. An in silico docking simulation and an in vitro HT universal colorimetric PARP assay confirmed that the VEGFR inhibitor Vatalanib exhibits off-target activity as a PARP inhibitor, broadening its mode of action. In contrast to the 2D-similarity search, the 3D-similarity landscape comparison identifies new functions and side effects of the known VEGFR inhibitor Vatalanib.

  1. Oenothera paradoxa defatted seeds extract and its bioactive component penta-O-galloyl-β-D-glucose decreased production of reactive oxygen species and inhibited release of leukotriene B4, interleukin-8, elastase, and myeloperoxidase in human neutrophils.

    PubMed

    Kiss, Anna K; Filipek, Agnieszka; Czerwińska, Monika; Naruszewicz, Marek

    2010-09-22

    In this study, we analyzed ex vivo the effect of an aqueous extract of Oenothera paradoxa defatted seeds on the formation of neutrophil-derived oxidants. For defining active compounds, we also tested lypophilic extract constituents such as gallic acid, (+)-catechin, ellagic acid, and penta-O-galloyl-β-D-glucose and a hydrophilic fraction containing polymeric procyanidins. The anti-inflammatory potential of the extract and compounds was tested by determining the release from activated neutrophils of elastase, myeloperoxidase, interleukin-8 (IL-8), and leukotriene B4 (LTB4), which are considered relevant for the pathogenesis of cardiovascular diseases. The extract of O. paradoxa defatted seeds displays potent antioxidant effects against both 4β-phorbol-12β-myristate-α13-acetate- and formyl-met-leu-phenylalanine-induced reactive oxygen species production in neutrophils with IC50 values around 0.2 μg/mL. All types of polyphenolics present in the extract contributed to the extract antioxidant activity. According to their IC50 values, penta-O-galloyl-β-D-glucose was the more potent constituent of the extract. In cell-free assays, we demonstrated that this effect is partially due to the scavenging of O2- and H2O2 oxygen species. The extract and especially penta-O-galloyl-β-D-glucose significantly inhibit elastase, myeloperoxidase IL-8, and LTB4 release with an IC50 for penta-O-galloyl-β-D-glucose of 17±1, 15±1, 6.5±2.5, and around 20 μM, respectively. The inhibition of penta-O-galloyl-β-D-glucose on reactive oxygen species and especially on O2- production, myeloperoxidase, and chemoattractant release may reduce the interaction of polymorphonuclear leukocyte with the vascular endothelium and by that potentially diminish the risk of progression of atherosclerosis development.

  2. An integrated structure- and system-based framework to identify new targets of metabolites and known drugs

    PubMed Central

    Naveed, Hammad; Hameed, Umar S.; Harrus, Deborah; Bourguet, William; Arold, Stefan T.; Gao, Xin

    2015-01-01

    Motivation: The inherent promiscuity of small molecules towards protein targets impedes our understanding of healthy versus diseased metabolism. This promiscuity also poses a challenge for the pharmaceutical industry as identifying all protein targets is important to assess (side) effects and repositioning opportunities for a drug. Results: Here, we present a novel integrated structure- and system-based approach of drug-target prediction (iDTP) to enable the large-scale discovery of new targets for small molecules, such as pharmaceutical drugs, co-factors and metabolites (collectively called ‘drugs’). For a given drug, our method uses sequence order–independent structure alignment, hierarchical clustering and probabilistic sequence similarity to construct a probabilistic pocket ensemble (PPE) that captures promiscuous structural features of different binding sites on known targets. A drug’s PPE is combined with an approximation of its delivery profile to reduce false positives. In our cross-validation study, we use iDTP to predict the known targets of 11 drugs, with 63% sensitivity and 81% specificity. We then predicted novel targets for these drugs—two that are of high pharmacological interest, the peroxisome proliferator-activated receptor gamma and the oncogene B-cell lymphoma 2, were successfully validated through in vitro binding experiments. Our method is broadly applicable for the prediction of protein-small molecule interactions with several novel applications to biological research and drug development. Availability and implementation: The program, datasets and results are freely available to academic users at http://sfb.kaust.edu.sa/Pages/Software.aspx. Contact: xin.gao@kaust.edu.sa and stefan.arold@kaust.edu.sa Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26286808

  3. Elevated plasma free fatty acids increase cardiovascular risk by inducing plasma biomarkers of endothelial activation, myeloperoxidase and PAI-1 in healthy subjects.

    PubMed

    Mathew, Manoj; Tay, Eric; Cusi, Kenneth

    2010-02-16

    CVD in obesity and T2DM are associated with endothelial activation, elevated plasma vascular inflammation markers and a prothrombotic state. We examined the contribution of FFA to these abnormalities following a 48-hour physiological increase in plasma FFA to levels of obesity and diabetes in a group of healthy subjects. 40 non-diabetic subjects (age = 38 +/- 3 yr, BMI = 28 +/- 1 kg/m2, FPG = 95 +/- 1 mg/dl, HbA1c = 5.3 +/- 0.1%) were admitted twice and received a 48-hour infusion of normal saline or low-dose lipid. Plasma was drawn for intracellular (ICAM-1) and vascular (VCAM-1) adhesion molecules-1, E-selectin (sE-S), myeloperoxidase (MPO) and total plasminogen inhibitor-1 (tPAI-1). Insulin sensitivity was measured by a hyperglycemic clamp (M/I). Lipid infusion increased plasma FFA to levels observed in obesity and T2DM and reduced insulin sensitivity by 27% (p = 0.01). Elevated plasma FFA increased plasma markers of endothelial activation ICAM-1 (138 +/- 10 vs. 186 +/- 25 ng/ml), VCAM-1 (1066 +/- 67 vs. 1204 +/- 65 ng/ml) and sE-S (20 +/- 1 vs. 24 +/- 1 ng/ml) between 13-35% and by > or = 2-fold plasma levels of myeloperoxidase (7.5 +/- 0.9 to 15 +/- 25 ng/ml), an inflammatory marker of future CVD, and tPAI-1 (9.7 +/- 0.6 to 22.5 +/- 1.5 ng/ml), an indicator of a prothrombotic state (all p < or = 0.01). The FFA-induced increase was independent from the degree of adiposity, being of similar magnitude in lean, overweight and obese subjects. An increase in plasma FFA within the physiological range observed in obesity and T2DM induces markers of endothelial activation, vascular inflammation and thrombosis in healthy subjects. This suggests that even transient (48-hour) and modest increases in plasma FFA may initiate early vascular abnormalities that promote atherosclerosis and CVD.

  4. Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer

    PubMed Central

    Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

    2016-01-01

    Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer “Big Data” has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of “hit” compounds. PMID:27806312

  5. Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

    PubMed Central

    Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen; Lyer, Stefan; Bewerunge-Hudler, Melanie; Gronert-Sum, Sabine; Strobel-Freidekind, Olga; Müller, Carolin; List, Markus; Jaskot, Aleksandra; Christiansen, Helle; Hafner, Mathias; Schadendorf, Dirk; Block, Ines; Mollenhauer, Jan

    2017-01-01

    Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression. PMID:28423600

  6. Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

    PubMed

    Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

    2017-07-01

    Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

  7. Systems level mapping of metabolic complexity in Mycobacterium tuberculosis to identify high-value drug targets.

    PubMed

    Vashisht, Rohit; Bhat, Ashwini G; Kushwaha, Shreeram; Bhardwaj, Anshu; Brahmachari, Samir K

    2014-10-11

    The effectiveness of current therapeutic regimens for Mycobacterium tuberculosis (Mtb) is diminished by the need for prolonged therapy and the rise of drug resistant/tolerant strains. This global health threat, despite decades of basic research and a wealth of legacy knowledge, is due to a lack of systems level understanding that can innovate the process of fast acting and high efficacy drug discovery. The enhanced functional annotations of the Mtb genome, which were previously obtained through a crowd sourcing approach was used to reconstruct the metabolic network of Mtb in a bottom up manner. We represent this information by developing a novel Systems Biology Spindle Map of Metabolism (SBSM) and comprehend its static and dynamic structure using various computational approaches based on simulation and design. The reconstructed metabolism of Mtb encompasses 961 metabolites, involved in 1152 reactions catalyzed by 890 protein coding genes, organized into 50 pathways. By accounting for static and dynamic analysis of SBSM in Mtb we identified various critical proteins required for the growth and survival of bacteria. Further, we assessed the potential of these proteins as putative drug targets that are fast acting and less toxic. Further, we formulate a novel concept of metabolic persister genes (MPGs) and compared our predictions with published in vitro and in vivo experimental evidence. Through such analyses, we report for the first time that de novo biosynthesis of NAD may give rise to bacterial persistence in Mtb under conditions of metabolic stress induced by conventional anti-tuberculosis therapy. We propose such MPG's as potential combination of drug targets for existing antibiotics that can improve their efficacy and efficiency for drug tolerant bacteria. The systems level framework formulated by us to identify potential non-toxic drug targets and strategies to circumvent the issue of bacterial persistence can substantially aid in the process of TB drug

  8. A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the Euprymna-Vibrio mutualism.

    PubMed

    Weis, V M; Small, A L; McFall-Ngai, M J

    1996-11-26

    Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts. It has been assumed that these specialized organs develop through a program of symbiosis-specific or -enhanced gene expression in one or both partners, but a clear example of this process has been lacking. In this study, we provide evidence for the enhanced production of an enzyme in the symbiotic organ of the squid Euprymna scolopes, which harbors a culture of the luminous bacterium Vibrio fischeri. Our data show that this enzyme has a striking biochemical similarity to mammalian myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine peroxidase that occurs in neutrophils. MPO and the squid peroxidase catalyze the same reaction, have similar apparent subunit molecular masses, and a polyclonal antibody to native human MPO specifically localized a peroxidase-like protein to the bacteria-containing regions of the symbiotic organ. We also provide evidence that a previously described squid cDNA encodes the protein (LO4) that is responsible for the observed dianisidine peroxidase activity. An antibody made against a fragment of LO4 immunoprecipiated dianisidine peroxidase activity from extracts of the symbiotic organ, and reacted against these extracts and human MPO in Western blot analysis. These data suggest that related biochemical mechanisms for the control of bacterial number and growth operate in associations that are as functionally diverse as pathogenesis and mutualism, and as phylogenetically distant as molluscs and mammals.

  9. An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2

    PubMed Central

    Boswell, C Andrew; Mundo, Eduardo E; Firestein, Ron; Zhang, Crystal; Mao, Weiguang; Gill, Herman; Young, Cynthia; Ljumanovic, Nina; Stainton, Shannon; Ulufatu, Sheila; Fourie, Aimee; Kozak, Katherine R; Fuji, Reina; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

    2013-01-01

    Background and Purpose The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. Experimental Approach A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography – X-ray computed tomography imaging and immunohistochemistry. Key Results The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Conclusions and Implications Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. PMID:22889168

  10. An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2.

    PubMed

    Boswell, C Andrew; Mundo, Eduardo E; Firestein, Ron; Zhang, Crystal; Mao, Weiguang; Gill, Herman; Young, Cynthia; Ljumanovic, Nina; Stainton, Shannon; Ulufatu, Sheila; Fourie, Aimee; Kozak, Katherine R; Fuji, Reina; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

    2013-01-01

    The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography - X-ray computed tomography imaging and immunohistochemistry. The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. © 2012 Genentech, Inc.. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  11. Can we use genetic and genomic approaches to identify candidate animals for targeted selective treatment.

    PubMed

    Laurenson, Yan C S M; Kyriazakis, Ilias; Bishop, Stephen C

    2013-10-18

    Estimated breeding values (EBV) for faecal egg count (FEC) and genetic markers for host resistance to nematodes may be used to identify resistant animals for selective breeding programmes. Similarly, targeted selective treatment (TST) requires the ability to identify the animals that will benefit most from anthelmintic treatment. A mathematical model was used to combine the concepts and evaluate the potential of using genetic-based methods to identify animals for a TST regime. EBVs obtained by genomic prediction were predicted to be the best determinant criterion for TST in terms of the impact on average empty body weight and average FEC, whereas pedigree-based EBVs for FEC were predicted to be marginally worse than using phenotypic FEC as a determinant criterion. Whilst each method has financial implications, if the identification of host resistance is incorporated into a wider genomic selection indices or selective breeding programmes, then genetic or genomic information may be plausibly included in TST regimes. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Manual method of visually identifying candidate signals for a targeted peptide.

    PubMed

    Filimonov, Aleksey; Kopylov, Arthur; Lisitsa, Andrey; Archakov, Alexander

    2018-04-15

    The purpose of this study is to improve peptide signal identification in groups of extracted ion chromatograms (XICs) obtained with the liquid chromatography-selected reaction monitoring (LC-SRM) technique and a triple quadrupole mass spectrometer (QqQ) operating in one of the supported multiple reaction monitoring (MRM) modes. The imperfection of quadrupole mass analyzers causes ion interference, which impedes the identification of peptide signals as chromatographic peak groups in relevant retention time intervals. To investigate this problem in depth, the QqQ conversion of the eluate into XIC groups was considered as the consecutive transformations of the particles' abundances as the corresponding functions of retention time. In this study, the hypothesis that, during this conversion, the same chromatographic profile should be preserved as an implicit sign in each chromatographic peak of the signal was confirmed for peptides. To examine chromatographic profiles, continuous transformations of XIC groups were derived and implemented in srm2prot Express software (s2pe, http://msr.ibmc.msk.ru/s2pe). Because of ion interference, several peptide-like signals may appear in one XIC group. Therefore, these signals must be considered candidates for a targeted peptide's signal and should be resolved after identification. The theoretical investigation of intensity functions as XICs that are not distorted by noise produced three rules for Identifying Candidate Signals for a targeted Peptide (ICSP, http://msr.ibmc.msk.ru/ICSP) that constitute the proposed manual visual method. We theoretically and experimentally compared this method with the conventional semiempirical intuitive technique and found that the former significantly streamlines peptide signal identification and avoids typical errors. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Measurement of myeloid maturation by flow cytochemistry in HL-60 leukemia: esterase is inducible, myeloperoxidase is not.

    PubMed

    Ross, D W

    1986-05-01

    The phenomenon of leukemic cell maturation requires a measurement of myeloid maturation to understand the process and to exploit it as a means of therapy for leukemia. The HL-60 leukemic cell line was used as a model of induced leukemic cell maturation in order to develop a method of quantitating granulocytic and monocytic maturation in response to drug therapy. An automated flow cytochemistry system (Hemalog-D) was employed to measure mean cell volume, myeloperoxidase (MPO), and nonspecific esterase (NSE). For granulocytic maturation induced by vitamin A or DMSO, MPO and cell volume decreased by 50%, maintaining a constant mean cellular MPO concentration throughout maturation from promyelocyte to neutrophil-like forms. For monocytic maturation induced by low-dose ARA-c, the mean NSE increased substantially, while cell volume remained constant. Unlike MPO concentration, NSE was truly inducible and thus a useful quantitative measure of maturation caused by low-dose ARA-c. Flow cytochemistry and cytofluorometry may be developed to allow for quantitative monitoring of therapeutic trials of induced maturation in human leukemias. However, this will require adapting these techniques to the complexity of human leukemias in vivo, and the necessity of handling heterogeneous populations encountered in bone marrow samples.

  14. Detection of anti-lactoferrin antibodies and anti-myeloperoxidase antibodies in autoimmune hepatitis: a retrospective study.

    PubMed

    Tan, Liming; Zhang, Yuhong; Peng, Weihua; Chen, Juanjuan; Li, Hua; Ming, Feng

    2014-01-01

    Anti-lactoferrin antibodies (ALA) and anti-myeloperoxidase antibodies (AMPA) are specific serological markers for autoimmune hepatitis (AIH). The project aimed to detect ALA and AMPA and explore their clinical significances in AIH patients. 59 AIH patients, 217 non AIH patients, and 50 healthy controls were enrolled in this study. ALA and AMPA were detected by ELISA. Antineutropil cytoplasmic antibodies (ANCA) and anti-smooth muscle antibodies (ASMA) were examined by indirect immunofluorescence. Antimitochondrial antibody M2 subtype (AMA-M2), anti-liver kidney microsomal antibody Type 1 (LKM1), anti-liver cytosol antibody Type 1 (LC1), and anti-soluble liver antigen/liver-pancreas antibodies (SLA/LP) were tested by immunoblot. The positivity for ALA was 18.6% in AIH group, only one patient in non-AIH group was positive for ALA; the positivity for AMPA was 59.3% in AIH group, with significant differences (P < 0.01) compared with other groups. The specificities for ALA and AMPA were 99.63% and 97.75%; the sensitivities were 18.64% and 59.32%; and the accuracy rates were 84.97% and 90.80%, respectively. A certain correlation was observed between ALA and SLA/LP, AMPA and ANCA, ASMA in AIH group. ALA and AMPA were associated with AIH, and had high clinical diagnostic value. Co-detection with other relative autoantibodies could play an important role in differential diagnosis of AIH.

  15. An Integrated Approach to Change the Outcome Part II: Targeted Neuromuscular Training Techniques to Reduce Identified ACL Injury Risk Factors

    PubMed Central

    Myer, Gregory D.; Ford, Kevin R.; Brent, Jensen L.; Hewett, Timothy E.

    2014-01-01

    Prior reports indicate that female athletes who demonstrate high knee abduction moments (KAMs) during landing are more responsive to neuromuscular training designed to reduce KAM. Identification of female athletes who demonstrate high KAM, which accurately identifies those at risk for noncontact anterior cruciate ligament (ACL) injury, may be ideal for targeted neuromuscular training. Specific neuromuscular training targeted to the underlying biomechanical components that increase KAM may provide the most efficient and effective training strategy to reduce noncontact ACL injury risk. The purpose of the current commentary is to provide an integrative approach to identify and target mechanistic underpinnings to increased ACL injury in female athletes. Specific neuromuscular training techniques will be presented that address individual algorithm components related to high knee load landing patterns. If these integrated techniques are employed on a widespread basis, prevention strategies for noncontact ACL injury among young female athletes may prove both more effective and efficient. PMID:22580980

  16. G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

    PubMed Central

    Silvescu, Cristina I.; Sackstein, Robert

    2014-01-01

    The host defense response critically depends on the production of leukocytes by the marrow and the controlled delivery of these cells to relevant sites of inflammation/infection. The cytokine granulocyte-colony stimulating factor (G-CSF) is commonly used therapeutically to augment neutrophil recovery following chemo/radiation therapy for malignancy, thereby decreasing infection risk. Although best known as a potent inducer of myelopoiesis, we previously reported that G-CSF also promotes the delivery of leukocytes to sites of inflammation by stimulating expression of potent E-selectin ligands, including an uncharacterized ∼65-kDa glycoprotein. To identify this ligand, we performed integrated biochemical analysis and mass spectrometry studies of G-CSF–treated primary human myeloid cells. Our studies show that this novel E-selectin ligand is a glycoform of the heavy chain component of the enzyme myeloperoxidase (MPO), a well-known lysosomal peroxidase. This specialized MPO glycovariant, referred to as “MPO–E-selectin ligand” (MPO–EL), is expressed on circulating G-CSF–mobilized leukocytes and is naturally expressed on blood myeloid cells in patients with febrile leukocytosis. In vitro biochemical studies show that G-CSF programs MPO–EL expression on human blood leukocytes and marrow myeloid cells via induction of N-linked sialofucosylations on MPO, with concomitant cell surface display of the molecule. MPO–EL is catalytically active and mediates angiotoxicity on human endothelial cells that express E-selectin. These findings thus define a G-CSF effect on MPO chemical biology that endows unsuspected functional versatility upon this enzyme, unveiling new perspectives on the biology of G-CSF and MPO, and on the role of E-selectin receptor/ligand interactions in leukocyte migration and vascular pathology. PMID:25002508

  17. High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines

    PubMed Central

    Cotesta, Simona; Perruccio, Francesca; Knapp, Britta; Fu, Yue; Studer, Christian; Pries, Verena; Riedl, Ralph; Helliwell, Stephen B.; Petrovic, Katarina T.; Movva, N. Rao; Sanglard, Dominique; Tao, Jianshi; Hoepfner, Dominic

    2016-01-01

    Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point. PMID:27855158

  18. Recurrent Targeted Genes of Hepatitis B Virus in the Liver Cancer Genomes Identified by a Next-Generation Sequencing–Based Approach

    PubMed Central

    Ding, Dong; Lou, Xiaoyan; Hua, Dasong; Yu, Wei; Li, Lisha; Wang, Jun; Gao, Feng; Zhao, Na; Ren, Guoping; Li, Lanjuan; Lin, Biaoyang

    2012-01-01

    Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent

  19. Rodent Models of Experimental Endometriosis: Identifying Mechanisms of Disease and Therapeutic Targets

    PubMed Central

    Bruner-Tran, Kaylon L.; Mokshagundam, Shilpa; Herington, Jennifer L.; Ding, Tianbing; Osteen, Kevin G.

    2018-01-01

    Background: Although it has been more than a century since endometriosis was initially described in the literature, understanding the etiology and natural history of the disease has been challenging. However, the broad utility of murine and rat models of experimental endometriosis has enabled the elucidation of a number of potentially targetable processes which may otherwise promote this disease. Objective: To review a variety of studies utilizing rodent models of endometriosis to illustrate their utility in examining mechanisms associated with development and progression of this disease. Results: Use of rodent models of endometriosis has provided a much broader understanding of the risk factors for the initial development of endometriosis, the cellular pathology of the disease and the identification of potential therapeutic targets. Conclusion: Although there are limitations with any animal model, the variety of experimental endometriosis models that have been developed has enabled investigation into numerous aspects of this disease. Thanks to these models, our under-standing of the early processes of disease development, the role of steroid responsiveness, inflammatory processes and the peritoneal environment has been advanced. More recent models have begun to shed light on how epigenetic alterations con-tribute to the molecular basis of this disease as well as the multiple comorbidities which plague many patients. Continued de-velopments of animal models which aid in unraveling the mechanisms of endometriosis development provide the best oppor-tunity to identify therapeutic strategies to prevent or regress this enigmatic disease.

  20. A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

    PubMed Central

    Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

    2014-01-01

    Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides. PMID:25084350

  1. Targeted and Untargeted Metabolic Profiling of Wild Grassland Plants identifies Antibiotic and Anthelmintic Compounds Targeting Pathogen Physiology, Metabolism and Reproduction.

    PubMed

    French, Katherine E; Harvey, Joe; McCullagh, James S O

    2018-01-26

    Plants traditionally used by farmers to manage livestock ailments could reduce reliance on synthetic antibiotics and anthelmintics but in many cases their chemical composition is unknown. As a case study, we analyzed the metabolite profiles of 17 plant species and 45 biomass samples from agricultural grasslands in England using targeted and untargeted metabolite profiling by liquid-chromatography mass spectrometry. We identified a range of plant secondary metabolites, including 32 compounds with known antimicrobial/anthelmintic properties which varied considerably across the different plant samples. These compounds have been shown previously to target multiple aspects of pathogen physiology and metabolism in vitro and in vivo, including inhibition of quorum sensing in bacteria and egg viability in nematodes. The most abundant bioactive compounds were benzoic acid, myricetin, p-coumaric acid, rhamnetin, and rosmarinic acid. Four wild plants (Filipendula ulmaria (L.) Maxim., Prunella vulgaris L., Centuarea nigra L., and Rhinanthus minor L.) and two forage legumes (Medicago sativa L., Trifolium hybridium L.) contained high levels of these compounds. Forage samples from native high-diversity grasslands had a greater abundance of medicinal compounds than samples from agriculturally improved grasslands. Incorporating plants with antibiotic/anthelmintic compounds into livestock feeds may reduce global drug-resistance and preserve the efficacy of last-resort drugs.

  2. BeReTa: a systematic method for identifying target transcriptional regulators to enhance microbial production of chemicals.

    PubMed

    Kim, Minsuk; Sun, Gwanggyu; Lee, Dong-Yup; Kim, Byung-Gee

    2017-01-01

    Modulation of regulatory circuits governing the metabolic processes is a crucial step for developing microbial cell factories. Despite the prevalence of in silico strain design algorithms, most of them are not capable of predicting required modifications in regulatory networks. Although a few algorithms may predict relevant targets for transcriptional regulator (TR) manipulations, they have limited reliability and applicability due to their high dependency on the availability of integrated metabolic/regulatory models. We present BeReTa (Beneficial Regulator Targeting), a new algorithm for prioritization of TR manipulation targets, which makes use of unintegrated network models. BeReTa identifies TR manipulation targets by evaluating regulatory strengths of interactions and beneficial effects of reactions, and subsequently assigning beneficial scores for the TRs. We demonstrate that BeReTa can predict both known and novel TR manipulation targets for enhanced production of various chemicals in Escherichia coli Furthermore, through a case study of antibiotics production in Streptomyces coelicolor, we successfully demonstrate its wide applicability to even less-studied organisms. To the best of our knowledge, BeReTa is the first strain design algorithm exclusively designed for predicting TR manipulation targets. MATLAB code is available at https://github.com/kms1041/BeReTa (github). byungkim@snu.ac.krSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Antineutrophil cytoplasmic autoantibodies and myeloperoxidase autoantibodies in clinical expression of Churg-Strauss syndrome.

    PubMed

    Healy, Bridget; Bibby, Susan; Steele, Richard; Weatherall, Mark; Nelson, Harold; Beasley, Richard

    2013-02-01

    The clinical significance of antineutrophil cytoplasmic antibodies (ANCAs) in the phenotypic expression of Churg-Strauss syndrome (CSS) is uncertain. We sought to investigate the relationship between ANCA status and the clinical expression of CSS in a case series derived from the US Food and Drug Administration's adverse events database. All cases of CSS reported to the US Food and Drug Administration from 1997 to April 2003 were reviewed. Information about basic demographics, suspect medication use, clinical manifestations, histologic findings, ANCA staining patterns, and the presence of antibodies to myeloperoxidase (anti-MPO) or proteinase 3 (anti-PR3) was recorded when available. There were 93 case reports of CSS with sufficient documentation, including ANCA status. There were 38 (40.9%) of 93 cases with positive ANCA results, of which 15 cases reported a positive ELISA, all of which were positive for anti-MPO. ANCA negativity was associated with an increased proportion of cardiac involvement (risk difference [RD], 38.2%; 95% CI, 25.3% to 51.0%), gastrointestinal involvement (RD, 25.5%; 95% CI, 13.9% to 37.0%), pulmonary infiltrates (odds ratio, 4.9; 95% CI, 1.5-16.2), and the outcome of a life-threatening event or death (RD, 30.9%; 95% CI, 18.7% to 43.1%) when compared with anti-MPO-positive cases. ANCA negativity was associated with a decreased proportion of peripheral neuropathy (odds ratio, 0.3; 95% CI, 0.07-0.9). These findings support the hypothesis that the presence or absence of autoantibodies influences the clinical expression and severity of CSS. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  4. Myeloperoxidase serves as a redox switch that regulates apoptosis in epithelial ovarian cancer.

    PubMed

    Saed, Ghassan M; Ali-Fehmi, Rouba; Jiang, Zhong L; Fletcher, Nicole M; Diamond, Michael P; Abu-Soud, Husam M; Munkarah, Adnan R

    2010-02-01

    Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO(+))-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO(+). We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT-PCR, ELISA, and TUNEL assays. MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions. Copyright 2009 Elsevier Inc. All rights reserved.

  5. A probabilistic approach to identify putative drug targets in biochemical networks.

    PubMed

    Murabito, Ettore; Smallbone, Kieran; Swinton, Jonathan; Westerhoff, Hans V; Steuer, Ralf

    2011-06-06

    Network-based drug design holds great promise in clinical research as a way to overcome the limitations of traditional approaches in the development of drugs with high efficacy and low toxicity. This novel strategy aims to study how a biochemical network as a whole, rather than its individual components, responds to specific perturbations in different physiological conditions. Proteins exerting little control over normal cells and larger control over altered cells may be considered as good candidates for drug targets. The application of network-based drug design would greatly benefit from using an explicit computational model describing the dynamics of the system under investigation. However, creating a fully characterized kinetic model is not an easy task, even for relatively small networks, as it is still significantly hampered by the lack of data about kinetic mechanisms and parameters values. Here, we propose a Monte Carlo approach to identify the differences between flux control profiles of a metabolic network in different physiological states, when information about the kinetics of the system is partially or totally missing. Based on experimentally accessible information on metabolic phenotypes, we develop a novel method to determine probabilistic differences in the flux control coefficients between the two observable phenotypes. Knowledge of how differences in flux control are distributed among the different enzymatic steps is exploited to identify points of fragility in one of the phenotypes. Using a prototypical cancerous phenotype as an example, we demonstrate how our approach can assist researchers in developing compounds with high efficacy and low toxicity. © 2010 The Royal Society

  6. A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

    PubMed Central

    Giotopoulos, George; van der Weyden, Louise; Osaki, Hikari; Rust, Alistair G.; Gallipoli, Paolo; Meduri, Eshwar; Horton, Sarah J.; Chan, Wai-In; Foster, Donna; Prinjha, Rab K.; Pimanda, John E.; Tenen, Daniel G.; Vassiliou, George S.; Koschmieder, Steffen; Adams, David J.

    2015-01-01

    The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. PMID:26304963

  7. Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

    PubMed Central

    Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

    2015-01-01

    Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID. PMID:26263206

  8. Extracellular matrix mediators of metastatic cell colonization characterized using scaffold mimics of the pre-metastatic niche.

    PubMed

    Aguado, Brian A; Caffe, Jordan R; Nanavati, Dhaval; Rao, Shreyas S; Bushnell, Grace G; Azarin, Samira M; Shea, Lonnie D

    2016-03-01

    Metastatic tumor cells colonize the pre-metastatic niche, which is a complex microenvironment consisting partially of extracellular matrix (ECM) proteins. We sought to identify and validate novel contributors to tumor cell colonization using ECM-coated poly(ε-caprolactone) (PCL) scaffolds as mimics of the pre-metastatic niche. Utilizing orthotopic breast cancer mouse models, fibronectin and collagen IV-coated scaffolds implanted in the subcutaneous space captured colonizing tumor cells, showing a greater than 2-fold increase in tumor cell accumulation at the implant site compared to uncoated scaffolds. As a strategy to identify additional ECM colonization contributors, decellularized matrix (DCM) from lungs and livers containing metastatic tumors were characterized. In vitro, metastatic cell adhesion was increased on DCM coatings from diseased organs relative to healthy DCM. Furthermore, in vivo implantations of diseased DCM-coated scaffolds had increased tumor cell colonization relative to healthy DCM coatings. Mass-spectrometry proteomics was performed on healthy and diseased DCM to identify candidates associated with colonization. Myeloperoxidase was identified as abundantly present in diseased organs and validated as a contributor to colonization using myeloperoxidase-coated scaffold implants. This work identified novel ECM proteins associated with colonization using decellularization and proteomics techniques and validated candidates using a scaffold to mimic the pre-metastatic niche. The pre-metastatic niche consists partially of ECM proteins that promote metastatic cell colonization to a target organ. We present a biomaterials-based approach to mimic this niche and identify ECM mediators of colonization. Using murine breast cancer models, we implanted microporous PCL scaffolds to recruit colonizing tumor cells in vivo. As a strategy to modulate colonization, we coated scaffolds with various ECM proteins, including decellularized lung and liver matrix from

  9. Myeloperoxidase and adenosine-deaminase levels in the pleural fluid leakage induced by carrageenan in the mouse model of pleurisy.

    PubMed Central

    Fröde, T S; Medeiros, Y S

    2001-01-01

    BACKGROUND: Although myeloperoxidase (MPO) and adenosine-deaminase (ADA) levels are markers of activated leukocytes, both enzymes have not been currently addressed in inflammation models. AIMS: This study evaluates whether the concentrations of these enzymes are significantly correlated with the content of leukocytes in a pleurisy model. METHODS: The pleurisy was induced by carrageenan (1%) in mice, and the parameters analyzed 4 and 48 h after. RESULTS: After the induction of inflammation (4h), MPO and ADA levels peaked in parallel to neutrophils (p<0.01). Regarding the second phase of pleurisy (48 h), the highest concentrations of ADA were detected in parallel to the highest levels of mononuclears (p<0.01). At this time, MPO levels and neutrophils remained elevated, although at lower levels than those found at 4 h. A significant positive correlation was found among neutrophiLs and MPO, and mononuclears and ADA (p<0.01). CONCLUSIONS: These findings support the evidence that both enzymes are markers of the inflammatory process, and provide new tools for a better understanding of the immunoregulatory pathways that occur in inflammation. PMID:11577999

  10. Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations.

    PubMed

    Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk; Griewank, Klaus G; Roesch, Alexander

    2017-06-20

    Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

  11. Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations

    PubMed Central

    Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk

    2017-01-01

    Purpose Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. Experimental Design and Results In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Conclusions Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors. PMID:28380455

  12. Targeted Metabolomics Identifies Pharmacodynamic Biomarkers for BIO 300 Mitigation of Radiation-Induced Lung Injury.

    PubMed

    Jones, Jace W; Jackson, Isabel L; Vujaskovic, Zeljko; Kaytor, Michael D; Kane, Maureen A

    2017-12-01

    Biomarkers serve a number of purposes during drug development including defining the natural history of injury/disease, serving as a secondary endpoint or trigger for intervention, and/or aiding in the selection of an effective dose in humans. BIO 300 is a patent-protected pharmaceutical formulation of nanoparticles of synthetic genistein being developed by Humanetics Corporation. The primary goal of this metabolomic discovery experiment was to identify biomarkers that correlate with radiation-induced lung injury and BIO 300 efficacy for mitigating tissue damage based upon the primary endpoint of survival. High-throughput targeted metabolomics of lung tissue from male C57L/J mice exposed to 12.5 Gy whole thorax lung irradiation, treated daily with 400 mg/kg BIO 300 for either 2 weeks or 6 weeks starting 24 h post radiation exposure, were assayed at 180 d post-radiation to identify potential biomarkers. A panel of lung metabolites that are responsive to radiation and able to distinguish an efficacious treatment schedule of BIO 300 from a non-efficacious treatment schedule in terms of 180 d survival were identified. These metabolites represent potential biomarkers that could be further validated for use in drug development of BIO 300 and in the translation of dose from animal to human.

  13. Salivary Myeloperoxidase, Assessed by 3,3'-Diaminobenzidine Colorimetry, Can Differentiate Periodontal Patients from Nonperiodontal Subjects.

    PubMed

    Klangprapan, Supaporn; Chaiyarit, Ponlatham; Hormdee, Doosadee; Kampichai, Amonrujee; Khampitak, Tueanjit; Daduang, Jureerut; Tavichakorntrakool, Ratree; Panijpan, Bhinyo; Boonsiri, Patcharee

    2016-01-01

    Periodontal diseases, which result from inflammation of tooth supporting tissues, are highly prevalent worldwide. Myeloperoxidase (MPO), from certain white blood cells in saliva, is a biomarker for inflammation. We report our study on the salivary MPO activity and its association with severity of periodontal diseases among Thai patients. Periodontally healthy subjects (n = 11) and gingivitis (n = 32) and periodontitis patients (n = 19) were enrolled. Assessments of clinically periodontal parameters were reported as percentages for gingival bleeding index (GI) and bleeding on probing (BOP), whereas pocket depth (PD) and clinical attachment loss (CAL) were measured in millimeters and then made to index scores. Salivary MPO activity was measured by colorimetry using 3,3'-diaminobenzidine as substrate. The results showed that salivary MPO activity in periodontitis patients was significantly higher than in healthy subjects (p = 0.003) and higher than in gingivitis patients (p = 0.059). No difference was found between gingivitis and healthy groups (p = 0.181). Significant correlations were observed (p < 0.01) between salivary MPO activity and GI (r = 0.632, p < 0.001), BOP (r = 0.599, p < 0.001), PD (r = 0.179, p = 0.164), and CAL (r = 0.357, p = 0.004) index scores. Sensitivity (94.12%), specificity (54.55%), and positive (90.57%) and negative (66.67%) predictive values indicate that salivary MPO activity has potential use as a screening marker for oral health of the Thai community.

  14. Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

    PubMed

    Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

    2012-12-01

    What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To

  15. siRNA screen identifies QPCT as a druggable target for Huntington's disease.

    PubMed

    Jimenez-Sanchez, Maria; Lam, Wun; Hannus, Michael; Sönnichsen, Birte; Imarisio, Sara; Fleming, Angeleen; Tarditi, Alessia; Menzies, Fiona; Dami, Teresa Ed; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O'Kane, Cahir J; Caricasole, Andrea; Rubinsztein, David C

    2015-05-01

    Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

  16. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic retinopathy.

    PubMed

    Sinha, Shivani; Saxena, Sandeep; Prasad, Senthamizh; Mahdi, Abbas Ali; Bhasker, Shashi Kumar; Das, Siddharth; Krasnik, Vladimir; Caprnda, Martin; Opatrilova, Radka; Kruzliak, Peter

    2017-05-01

    To study the association of serum levels of anti-myeloperoxidase (MPO) antibody with retinal photoreceptor ellipsoid zone (EZ) disruption in diabetic retinopathy. Consecutive patients with type 2 DM [diabetes mellitus with no retinopathy (NODR; n=20); non-proliferative diabetic retinopathy (NPDR; n=18); proliferative diabetic retinopathy (PDR; n=16)] and healthy controls (n=20) between the ages of 40 and 65years were included. Disruption of EZ was graded by spectral domain optical coherence tomography as no disruption of EZ and disrupted EZ. The serum levels of anti-MPO antibody was analyzed using standard protocol. Association between the variables was evaluated using multiple regression analysis. A significant difference was found between the serum levels of anti-MPO antibody in various study groups (p<0.001). A positive association was found between EZ disruption and levels of anti-MPO antibody [adjusted odd's ratio (AOR)=1.079, CI 1.010-1.124, p=0.04]. A significant positive correlation was found between logMAR visual acuity and grade of disruption (AOR=1.008, CI 1.006-5.688, p=0.04). An increased serum anti-MPO antibody levels is associated with retinal photoreceptor EZ disruption and decreased visual acuity in diabetic retinopathy. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs.

    PubMed

    Noh, Jaeduk Yoshimura; Yasuda, Shigemitu; Sato, Shotaro; Matsumoto, Masako; Kunii, Yo; Noguchi, Yoshihiko; Mukasa, Koji; Ito, Kunihiko; Ito, Koichi; Sugiyama, Osamu; Kobayashi, Hiroshi; Nihojima, Shigeru; Okazaki, Masaru; Yokoyama, Shunji

    2009-08-01

    The clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis caused by antithyroid drugs are still unclear because most reports describe only a small number of patients. The objective was to analyze a large number of patients with MPO-ANCA-associated vasculitis to determine the time of onset, the drug and dose taken, the clinical symptoms, the relationship between the clinical symptoms and the MPO-ANCA titer, and the incidence. We analyzed 92 patients in whom the adverse reaction of MPO-ANCA-associated vasculitis was reported to Chugai Pharmaceutical, a company that markets antithyroid drugs. Of the 92 patients, 41 (44.6%) had single-organ failure, 32 (34.8%) had two-organ failure, 13 (14.1%), had three-organ failure, and two (2.2%) had four-organ failure. The number of organs involved was unknown in the other four patients (4.3%). The median time of onset was 42 months (range, 1-372 months) after starting drug treatment. The median dose at onset of MPO-ANCA-associated vasculitis was 15 mg/d (range, 2.5-45 mg/d) for methimazole and 200 mg/d (50-450 mg/d) for propylthiouracil. The severity and number of organs involved were not correlated with the MPO-ANCA titer. The incidence was between 0.53 and 0.79 patients per 10,000, and the ratio of the estimated incidences for methimazole and propylthiouracil was 1:39.2. The time of onset of MPO-ANCA-associated vasculitis and the dose at onset varied. The severity and number of organs involved were not correlated with the MPO-ANCA titer, indicating a need for vigilance even when the MPO-ANCA titer is only weakly positive.

  18. A Screening of UNF Targets Identifies Rnb, a Novel Regulator of Drosophila Circadian Rhythms.

    PubMed

    Kozlov, Anatoly; Jaumouillé, Edouard; Machado Almeida, Pedro; Koch, Rafael; Rodriguez, Joseph; Abruzzi, Katharine C; Nagoshi, Emi

    2017-07-12

    Behavioral circadian rhythms are controlled by multioscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions (Lee et al., 2016; Top et al., 2016). The nuclear receptor unfulfilled ( unf ) represents a regulatory node that provides the small ventral lateral neurons (s-LNvs) unique characteristics as the master pacemaker (Beuchle et al., 2012). We previously showed that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period ( per ) (Jaumouillé et al., 2015). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, we identified UNF target genes using chromatin immunoprecipitation. Our data demonstrate that a previously uncharacterized gene CG7837 , which we termed R and B ( Rnb ), acts downstream of UNF to regulate the function of the s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus, and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit. SIGNIFICANCE STATEMENT Circadian behavior is generated by a pacemaker circuit comprising diverse classes of pacemaker neurons, each of which contains a molecular clock. In addition to the anatomical and functional diversity, recent studies have shown the mechanistic differences in the molecular clockwork among the pacemaker neurons in Drosophila Here, we identified the molecular characteristics

  19. A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of α1-antitrypsin deficiency.

    PubMed

    O'Reilly, Linda P; Long, Olivia S; Cobanoglu, Murat C; Benson, Joshua A; Luke, Cliff J; Miedel, Mark T; Hale, Pamela; Perlmutter, David H; Bahar, Ivet; Silverman, Gary A; Pak, Stephen C

    2014-10-01

    α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog-ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models. | Office of Cancer Genomics

    Cancer.gov

    T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs).

  1. Open Targets: a platform for therapeutic target identification and validation

    PubMed Central

    Koscielny, Gautier; An, Peter; Carvalho-Silva, Denise; Cham, Jennifer A.; Fumis, Luca; Gasparyan, Rippa; Hasan, Samiul; Karamanis, Nikiforos; Maguire, Michael; Papa, Eliseo; Pierleoni, Andrea; Pignatelli, Miguel; Platt, Theo; Rowland, Francis; Wankar, Priyanka; Bento, A. Patrícia; Burdett, Tony; Fabregat, Antonio; Forbes, Simon; Gaulton, Anna; Gonzalez, Cristina Yenyxe; Hermjakob, Henning; Hersey, Anne; Jupe, Steven; Kafkas, Şenay; Keays, Maria; Leroy, Catherine; Lopez, Francisco-Javier; Magarinos, Maria Paula; Malone, James; McEntyre, Johanna; Munoz-Pomer Fuentes, Alfonso; O'Donovan, Claire; Papatheodorou, Irene; Parkinson, Helen; Palka, Barbara; Paschall, Justin; Petryszak, Robert; Pratanwanich, Naruemon; Sarntivijal, Sirarat; Saunders, Gary; Sidiropoulos, Konstantinos; Smith, Thomas; Sondka, Zbyslaw; Stegle, Oliver; Tang, Y. Amy; Turner, Edward; Vaughan, Brendan; Vrousgou, Olga; Watkins, Xavier; Martin, Maria-Jesus; Sanseau, Philippe; Vamathevan, Jessica; Birney, Ewan; Barrett, Jeffrey; Dunham, Ian

    2017-01-01

    We have designed and developed a data integration and visualization platform that provides evidence about the association of known and potential drug targets with diseases. The platform is designed to support identification and prioritization of biological targets for follow-up. Each drug target is linked to a disease using integrated genome-wide data from a broad range of data sources. The platform provides either a target-centric workflow to identify diseases that may be associated with a specific target, or a disease-centric workflow to identify targets that may be associated with a specific disease. Users can easily transition between these target- and disease-centric workflows. The Open Targets Validation Platform is accessible at https://www.targetvalidation.org. PMID:27899665

  2. Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

    PubMed Central

    Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

    2015-01-01

    Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

  3. Rhodium(II) proximity-labeling identifies a novel target site on STAT3 for inhibitors with potent anti-leukemia activity

    PubMed Central

    Minus, Matthew B.; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M.; Long, Xin; Krueger, Michael; Stevens, Alexandra; Kolosov, Mikhail I.; Sison, Edward Allen R.; Ball, Zachary T.

    2015-01-01

    Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. PMID:26480340

  4. Identifying initial molecular targets of PDT: protein and lipid oxidation products

    NASA Astrophysics Data System (ADS)

    Oleinick, Nancy L.; Kim, Junhwan; Rodriguez, Myriam E.; Xue, Liang-yan; Kenney, Malcolm E.; Anderson, Vernon E.

    2009-06-01

    Photodynamic Therapy (PDT) generates singlet oxygen (1O2) which oxidizes biomolecules in the immediate vicinity of its formation. The phthalocyanine photosensitizer Pc 4 localizes to mitochondria and endoplasmic reticulum, and the primary targets of Pc 4-PDT are expected to be lipids and proteins of those membranes. The initial damage then causes apoptosis in cancer cells via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by the activation of caspases. That damage also triggers the induction of autophagy, an attempt by the cells to eliminate damaged organelles, or when damage is too extensive, to promote cell death. Cyt-c is bound to the cytosolic side of the mitochondrial inner membrane through association with cardiolipin (CL), a phospholipid containing four unsaturated fatty acids and thus easily oxidized by 1O2 or by other oxidizing agents. Increasing evidence suggests that oxidation of CL loosens its association with Cyt-c, and that the peroxidase activity of Cyt-c can oxidize CL. In earlier studies of Cyt-c in homogeneous medium by MALDI-TOF-MS and LC-ESI-MS, we showed that 1O2 generated by Pc 4-PDT oxidized histidine, methionine, tryptophan, and unexpectedly phenylalanine but not tyrosine. Most of the oxidation products were known to be formed by other oxidizing agents, such as hydroxyl radical, superoxide radical anion, and peroxynitrite. However, two products of histidine were unique to 1O2 and may be useful for reporting the action of 1O2 in cells and tissues. These products, as well as CL oxidation products, have now been identified in liposomes and mitochondria after Pc 4-PDT. In mitochondria, the PDT dose-dependent oxidations can be related to specific changes in mitochondrial function, Bcl-2 photodamage, and Cyt-c release. Thus, the role of PDT-generated 1O2 in oxidizing Cyt-c and CL and the interplay between protein and lipid targets may be highly relevant to understanding one mechanism for cell killing by PDT.

  5. Combining a nontargeted and targeted metabolomics approach to identify metabolic pathways significantly altered in polycystic ovary syndrome.

    PubMed

    Chang, Alice Y; Lalia, Antigoni Z; Jenkins, Gregory D; Dutta, Tumpa; Carter, Rickey E; Singh, Ravinder J; Nair, K Sreekumaran

    2017-06-01

    Polycystic ovary syndrome (PCOS) is a condition of androgen excess and chronic anovulation frequently associated with insulin resistance. We combined a nontargeted and targeted metabolomics approach to identify pathways and metabolites that distinguished PCOS from metabolic syndrome (MetS). Twenty obese women with PCOS were compared with 18 obese women without PCOS. Both groups met criteria for MetS but could not have diabetes mellitus or take medications that treat PCOS or affect lipids or insulin sensitivity. Insulin sensitivity was derived from the frequently sampled intravenous glucose tolerance test. A nontargeted metabolomics approach was performed on fasting plasma samples to identify differentially expressed metabolites, which were further evaluated by principal component and pathway enrichment analysis. Quantitative targeted metabolomics was then applied on candidate metabolites. Measured metabolites were tested for associations with PCOS and clinical variables by logistic and linear regression analyses. This multiethnic, obese sample was matched by age (PCOS, 37±6; MetS, 40±6years) and body mass index (BMI) (PCOS, 34.6±5.1; MetS, 33.7±5.2kg/m 2 ). Principal component analysis of the nontargeted metabolomics data showed distinct group separation of PCOS from MetS controls. From the subset of 385 differentially expressed metabolites, 22% were identified by accurate mass, resulting in 19 canonical pathways significantly altered in PCOS, including amino acid, lipid, steroid, carbohydrate, and vitamin D metabolism. Targeted metabolomics identified many essential amino acids, including branched-chain amino acids (BCAA) that were elevated in PCOS compared with MetS. PCOS was most associated with BCAA (P=.02), essential amino acids (P=.03), the essential amino acid lysine (P=.02), and the lysine metabolite α-aminoadipic acid (P=.02) in models adjusted for surrogate variables representing technical variation in metabolites. No significant differences between

  6. Combining a Nontargeted and Targeted Metabolomics Approach to Identify Metabolic Pathways Significantly Altered in Polycystic Ovary Syndrome

    PubMed Central

    Chang, Alice Y.; Lalia, Antigoni Z.; Jenkins, Gregory D.; Dutta, Tumpa; Carter, Rickey E.; Singh, Ravinder J.; Sreekumaran Nair, K.

    2017-01-01

    Objective Polycystic ovary syndrome (PCOS) is a condition of androgen excess and chronic anovulation frequently associated with insulin resistance. We combined a nontargeted and targeted metabolomics approach to identify pathways and metabolites that distinguished PCOS from metabolic syndrome (MetS). Methods Twenty obese women with PCOS were compared with 18 obese women without PCOS. Both groups met criteria for MetS but could not have diabetes mellitus or take medications that treat PCOS or affect lipids or insulin sensitivity. Insulin sensitivity was derived from the frequently sampled intravenous glucose tolerance test. A nontargeted metabolomics approach was performed on fasting plasma samples to identify differentially expressed metabolites, which were further evaluated by principal component and pathway enrichment analysis. Quantitative targeted metabolomics was then applied on candidate metabolites. Measured metabolites were tested for associations with PCOS and clinical variables by logistic and linear regression analyses. Results This multiethnic, obese sample was matched by age (PCOS, 37 ± 6; MetS, 40 ± 6 years) and body mass index (BMI) (PCOS, 34.6 ± 5.1; MetS, 33.7 ± 5.2 kg/m2). Principal component analysis of the nontargeted metabolomics data showed distinct group separation of PCOS from MetS controls. From the subset of 385 differentially expressed metabolites, 22% were identified by accurate mass, resulting in 19 canonical pathways significantly altered in PCOS, including amino acid, lipid, steroid, carbohydrate, and vitamin D metabolism. Targeted metabolomics identified many essential amino acids, including branched-chain amino acids (BCAA) that were elevated in PCOS compared with MetS. PCOS was most associated with BCAA (P = .02), essential amino acids (P = .03), the essential amino acid lysine (P = .02), and the lysine metabolite α-aminoadipic acid (P = .02) in models adjusted for surrogate variables representing technical variation in

  7. Soft computing model for optimized siRNA design by identifying off target possibilities using artificial neural network model.

    PubMed

    Murali, Reena; John, Philips George; Peter S, David

    2015-05-15

    The ability of small interfering RNA (siRNA) to do posttranscriptional gene regulation by knocking down targeted genes is an important research topic in functional genomics, biomedical research and in cancer therapeutics. Many tools had been developed to design exogenous siRNA with high experimental inhibition. Even though considerable amount of work has been done in designing exogenous siRNA, design of effective siRNA sequences is still a challenging work because the target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. In some cases, siRNAs may tolerate mismatches with the target mRNA, but knockdown of genes other than the intended target could make serious consequences. Hence to design siRNAs, two important concepts must be considered: the ability in knocking down target genes and the off target possibility on any nontarget genes. So before doing gene silencing by siRNAs, it is essential to analyze their off target effects in addition to their inhibition efficacy against a particular target. Only a few methods have been developed by considering both efficacy and off target possibility of siRNA against a gene. In this paper we present a new design of neural network model with whole stacking energy (ΔG) that enables to identify the efficacy and off target effect of siRNAs against target genes. The tool lists all siRNAs against a particular target with their inhibition efficacy and number of matches or sequence similarity with other genes in the database. We could achieve an excellent performance of Pearson Correlation Coefficient (R=0. 74) and Area Under Curve (AUC=0.906) when the threshold of whole stacking energy is ≥-34.6 kcal/mol. To the best of the author's knowledge, this is one of the best score while considering the "combined efficacy and off target possibility" of siRNA for silencing a gene. The proposed model

  8. Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

    PubMed Central

    2012-01-01

    Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the

  9. Monocyte-directed RNAi targeting CCR2 improves infarct healing in atherosclerosis-prone mice

    PubMed Central

    Majmudar, Maulik D.; Keliher, Edmund J.; Heidt, Timo; Leuschner, Florian; Truelove, Jessica; Sena, Brena F.; Gorbatov, Rostic; Iwamoto, Yoshiko; Dutta, Partha; Wojtkiewicz, Gregory; Courties, Gabriel; Sebas, Matt; Borodovsky, Anna; Fitzgerald, Kevin; Nolte, Marc W.; Dickneite, Gerhard; Chen, John W.; Anderson, Daniel G.; Swirski, Filip K.; Weissleder, Ralph; Nahrendorf, Matthias

    2013-01-01

    Background Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. We here employed nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apoE−/− mice after MI. We used dual target PET/MRI of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset-targeted RNAi altered infarct inflammation and healing. Methods and Results Flow cytometry, gene expression analysis and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix crosslinking non-invasively, we developed a fluorine-18 labeled PET agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged using a molecular MRI sensor of MPO activity (MPO-Gd). PET/MRI detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal, p<0.05) while 18F-FXIII PET reflected unimpeded matrix crosslinking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29 to 35% (p<0.05). Conclusion CCR2 targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation and curbed post-MI left ventricular remodeling. PMID:23616627

  10. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

    PubMed Central

    Ismail, Hanafy M.; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

    2016-01-01

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  11. Multidimensional Targeting: Identifying Beneficiaries of Conditional Cash Transfer Programs

    ERIC Educational Resources Information Center

    Azevedo, Viviane; Robles, Marcos

    2013-01-01

    Conditional cash transfer programs (CCTs) have two main objectives: reducing poverty and increasing the human capital of children. To reach these objectives, transfers are given to poor households conditioned on investments in their children's education, health, and nutrition. Targeting mechanisms used by CCTs have been generally successful in…

  12. Genome-Wide Association Study with Targeted and Non-targeted NMR Metabolomics Identifies 15 Novel Loci of Urinary Human Metabolic Individuality

    PubMed Central

    Raffler, Johannes; Friedrich, Nele; Arnold, Matthias; Kacprowski, Tim; Rueedi, Rico; Altmaier, Elisabeth; Bergmann, Sven; Budde, Kathrin; Gieger, Christian; Homuth, Georg; Pietzner, Maik; Römisch-Margl, Werner; Strauch, Konstantin; Völzke, Henry; Waldenberger, Melanie; Wallaschofski, Henri; Nauck, Matthias; Völker, Uwe; Kastenmüller, Gabi; Suhre, Karsten

    2015-01-01

    Genome-wide association studies with metabolic traits (mGWAS) uncovered many genetic variants that influence human metabolism. These genetically influenced metabotypes (GIMs) contribute to our metabolic individuality, our capacity to respond to environmental challenges, and our susceptibility to specific diseases. While metabolic homeostasis in blood is a well investigated topic in large mGWAS with over 150 known loci, metabolic detoxification through urinary excretion has only been addressed by few small mGWAS with only 11 associated loci so far. Here we report the largest mGWAS to date, combining targeted and non-targeted 1H NMR analysis of urine samples from 3,861 participants of the SHIP-0 cohort and 1,691 subjects of the KORA F4 cohort. We identified and replicated 22 loci with significant associations with urinary traits, 15 of which are new (HIBCH, CPS1, AGXT, XYLB, TKT, ETNPPL, SLC6A19, DMGDH, SLC36A2, GLDC, SLC6A13, ACSM3, SLC5A11, PNMT, SLC13A3). Two-thirds of the urinary loci also have a metabolite association in blood. For all but one of the 6 loci where significant associations target the same metabolite in blood and urine, the genetic effects have the same direction in both fluids. In contrast, for the SLC5A11 locus, we found increased levels of myo-inositol in urine whereas mGWAS in blood reported decreased levels for the same genetic variant. This might indicate less effective re-absorption of myo-inositol in the kidneys of carriers. In summary, our study more than doubles the number of known loci that influence urinary phenotypes. It thus allows novel insights into the relationship between blood homeostasis and its regulation through excretion. The newly discovered loci also include variants previously linked to chronic kidney disease (CPS1, SLC6A13), pulmonary hypertension (CPS1), and ischemic stroke (XYLB). By establishing connections from gene to disease via metabolic traits our results provide novel hypotheses about molecular mechanisms

  13. A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

    PubMed Central

    Bramsen, Jesper B.; Pakula, Malgorzata M.; Hansen, Thomas B.; Bus, Claus; Langkjær, Niels; Odadzic, Dalibor; Smicius, Romualdas; Wengel, Suzy L.; Chattopadhyaya, Jyoti; Engels, Joachim W.; Herdewijn, Piet; Wengel, Jesper; Kjems, Jørgen

    2010-01-01

    Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low. PMID:20453030

  14. Tandem application of ligand-based virtual screening and G4-OAS assay to identify novel G-quadruplex-targeting chemotypes.

    PubMed

    Musumeci, Domenica; Amato, Jussara; Zizza, Pasquale; Platella, Chiara; Cosconati, Sandro; Cingolani, Chiara; Biroccio, Annamaria; Novellino, Ettore; Randazzo, Antonio; Giancola, Concetta; Pagano, Bruno; Montesarchio, Daniela

    2017-05-01

    G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MTT assays. A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4-forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells. The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands. Expanding the repertoire of the available G4-targeting chemotypes with improved physico-chemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Enhanced detection of myeloperoxidase activity in deep tissues through luminescent excitation of near-infrared nanoparticles.

    PubMed

    Zhang, Ning; Francis, Kevin P; Prakash, Arun; Ansaldi, Daniel

    2013-04-01

    A previous study reported the use of luminol for the detection of myeloperoxidase (MPO) activity using optical imaging in infiltrating neutrophils under inflammatory disease conditions. The detection is based on a photon-emitting reaction between luminol and an MPO metabolite. Because of tissue absorption and scattering, however, luminol-emitted blue light can be efficiently detected from superficial inflammatory foci only. In this study we report a chemiluminescence resonance energy transfer (CRET) methodology in which luminol-generated blue light excites nanoparticles to emit light in the near-infrared spectral range, resulting in remarkable improvement of MPO detectability in vivo. CRET caused a 37-fold increase in luminescence emission over luminol alone in detecting MPO activity in lung tissues after lipopolysaccharide challenge. We demonstrated a dependence of the chemiluminescent signal on MPO activity using MPO-deficient mice. In addition, co-administration of 4-aminobenzoic acid hydrazide (4-ABAH), an irreversible inhibitor of MPO, significantly attenuated luminescent emission from inflamed lungs. Inhibition of nitric oxide synthase with a nonspecific inhibitor, L-NAME, had no effect on luminol-mediated chemiluminescence production. Pretreatment of mice with MLN120B, a selective inhibitor of IKK-2, resulted in suppression of neutrophil infiltration to the lung tissues and reduction of MPO activity. We also demonstrated that CRET can effectively detect MPO activity at deep tissue tumor foci due to tumor development-associated neutrophil infiltration. We developed a sensitive MPO detection methodology that provides a means for visualizing and quantifying oxidative stress in deep tissue. This method is amenable to rapid evaluation of anti-inflammatory agents in animal models.

  16. Usefulness of Myeloperoxidase Levels in Healthy Elderly Subjects to Predict Risk of Developing Heart Failure

    PubMed Central

    Tang, W.H. Wilson; Katz, Ronit; Brennan, Marie-Luise; Aviles, Ronnier J.; Tracy, Russell P.; Psaty, Bruce M.; Hazen, Stanley L.

    2009-01-01

    Increased systemic myeloperoxidase (MPO) has been associated with both the presence and severity of heart failure (HF). This study tested the hypothesis that increased systemic MPO in apparently healthy elderly subjects may predict increased risk of developing HF. Systemic MPO was measured in all available samples from the 1992 to 1993 visit of the Cardiovascular Health Study (CHS). After excluding subjects without available blood samples or with a history of prevalent HF, myocardial infarction (MI), or stroke, 3,733 subjects were included. A total of 569 subjects developed incident HF during 7.2 ± 2.3 years of follow-up. Patients in the highest MPO quartile (>432 pmol/L) showed higher risk of developing incident HF after adjusting for MI, age, gender, systolic blood pressure, smoking, low-density lipoprotein cholesterol, diabetes mellitus, and any subclinical cardiovascular disease (hazard ratio 1.34, 95% confidence interval 1.06 to 1.72, p = 0.013). However, the relation was more apparent after censoring subjects with incident MI before incident HF, even when adjusted for C-reactive protein and cystatin C (hazard ratio 1.46, 95% confidence interval 1.08 to 1.97, p = 0.02). Interestingly, stratified analyses showed that the relation between increased MPO and HF risk was stronger in subjects without traditional cardiovascular risk factors (≤75 years old, systolic blood pressure ≤136 mm Hg, no subclinical cardiovascular disease, and no diabetes mellitus). In conclusion, an independent association between increased MPO and the development of HF in apparently healthy elderly subjects was observed, particularly beyond MI and traditional cardiac risk factors. PMID:19406270

  17. Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction

    PubMed Central

    Art, Tatiana; Franck, Thierry; Lekeux, Pierre; de Moffarts, Brieuc; Couëtil, Laurent; Becker, Martine; Kohnen, Serge; Deby-Dupont, Ginette; Serteyn, Didier

    2006-01-01

    The aim of this work was to measure the myeloperoxidase (MPO) concentration in bronchoalveolar lavage (BAL) fluid collected from horses with recurrent airway obstruction (RAO), both in crisis and in remission, as well as from healthy horses. Seven horses with RAO were exposed to moldy hay until the maximum change in pleural pressure was greater than 1.5 kPa. At that point, BAL was performed, and the total cell counts and percentages in the fluid were immediately determined. To measure the MPO concentration in BAL-fluid supernatant, we used a specific enzyme-linked immunosorbent assay with polyclonal antibodies against equine MPO. The tests were repeated on the horses with RAO after they had spent 2 mo on pasture. Six healthy horses serving as controls underwent the same tests. The absolute and relative neutrophil counts and the MPO concentration in the BAL fluid were significantly greater in the horses with an RAO crisis than in the control horses. After 2 mo on pasture, the horses that had been in RAO crisis were clinically normal, and their neutrophil counts and MPO levels in BAL fluid had significantly decreased; during remission their neutrophil counts were not significantly different from those in the healthy horses, but their MPO concentration remained significantly higher. This study showed that determining the MPO concentration in a horse’s BAL fluid is technically possible and that during remission from RAO the concentration remains higher than normal. Thus, MPO may be a marker of neutrophil presence and activation in the lower airways. PMID:17042382

  18. Myeloperoxidase mediated HDL oxidation and HDL proteome changes do not contribute to dysfunctional HDL in Chinese subjects with coronary artery disease

    PubMed Central

    Yu, Haiyi; Li, Lei; He, Liyun; Gao, Wei; Liu, Xiaodan; Guo, Yanhong; Byun, Jaeman; Zhang, Jifeng; Chen, Y. Eugene

    2018-01-01

    High density lipoprotein (HDL) cholesterol levels and cholesterol efflux capacity (CEC) are inversely correlated with coronary artery disease (CAD) risk. Myeloperoxidase (MPO) derived oxidants and HDL proteome changes are implicated in HDL dysfunction in subjects with CAD in the United States; however, the effect of MPO on HDL function and HDL proteome in ethnic Chinese population is unknown. We recruited four matched ethnic Chinese groups (20 patients each): subjects with 1) low HDL levels (HDL levels in men <40mg/dL and women <50mg/dL) and non-CAD (identified by coronary angiography or cardiac CT angiography); 2) low HDL and CAD; 3) high HDL (men >50mg/dL; women >60mg/dL) with no CAD; and 4) high HDL with CAD. Serum cytokines, serum MPO levels, serum CEC, MPO-oxidized HDL tyrosine moieties, and HDL proteome were assessed by mass spectrometry individually in the four groups. The cytokines, MPO levels, and HDL proteome profiles were not significantly different between the four groups. As expected, CEC was depressed in the entire CAD group but more specifically in the CAD low-HDL group. HDL of CAD subjects had significantly higher 3-nitrotyrosine than non-CAD subjects, but the MPO-specific 3-chlorotyrosine was unchanged; CEC in the CAD low-HDL group did not correlate with either HDL 3-chlorotyrosine or 3-nitrotyrosine levels. Neither 3-chlorotyrosine, which is MPO-specific, nor 3-nitrotyrosine generated from MPO or other reactive nitrogen species was associated with CEC. MPO mediated oxidative stress and HDL proteome composition changes are not the primary cause HDL dysfunction in Chinese subjects with CAD. These studies highlight ethnic differences in HDL dysfunction between United States and Chinese cohorts raising possibility of unique pathways of HDL dysfunction in this cohort. PMID:29505607

  19. Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

    PubMed

    Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

    2015-10-26

    Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Preclinical studies identify novel targeted pharmacological strategies for treatment of human malignant pleural mesothelioma.

    PubMed

    Favoni, Roberto E; Daga, Antonio; Malatesta, Paolo; Florio, Tullio

    2012-05-01

    The incidence of human malignant pleural mesothelioma (hMPM) is still increasing worldwide. hMPM prognosis is poor even if the median survival time has been slightly improved after the introduction of the up-to-date chemotherapy. Nevertheless, large phase II/III trials support the combination of platinum derivatives and pemetrexed or raltitrexed, as preferred first-line schedule. Better understanding of the molecular machinery of hMPM will lead to the design and synthesis of novel compounds targeted against pathways identified as crucial for hMPM cell proliferation and spreading. Among them, several receptors tyrosine kinase show altered activity in subsets of hMPM. This observation suggests that these kinases might represent novel therapeutic targets in this chemotherapy-resistant disease. Over these foundations, several promising studies are ongoing at preclinical level and novel molecules are currently under evaluation as well. Yet, established tumour cell lines, used for decades to investigate the efficacy of anticancer agents, although still the main source of drug efficacy studies, after long-term cultures tend to biologically diverge from the original tumour, limiting the predictive potential of in vivo efficacy. Cancer stem cells (CSCs), a subpopulation of malignant cells capable of self-renewal and multilineage differentiation, are believed to play an essential role in cancer initiation, growth, metastasization and relapse, being responsible of chemo- and radiotherapy refractoriness. According to the current carcinogenesis theory, CSCs represent the tumour-initiating cell (TIC) fraction, the only clonogenic subpopulation able to originate a tumour mass. Consequently, the recently described isolation of TICs from hMPM, the proposed main pharmacological target for novel antitumoural drugs, may contribute to better dissect the biology and multidrug resistance pathways controlling hMPM growth. © 2012 The Authors. British Journal of Pharmacology © 2012 The

  1. Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia

    PubMed Central

    Geng, Huimin; Brennan, Sarah; Milne, Thomas A.; Chen, Wei-Yi; Li, Yushan; Hurtz, Christian; Kweon, Soo-Mi; Zickl, Lynette; Shojaee, Seyedmehdi; Neuberg, Donna; Huang, Chuanxin; Biswas, Debabrata; Xin, Yuan; Racevskis, Janis; Ketterling, Rhett P.; Luger, Selina M.; Lazarus, Hillard; Tallman, Martin S.; Rowe, Jacob M.; Litzow, Mark R.; Guzman, Monica L.; Allis, C. David; Roeder, Robert G.; Müschen, Markus; Paietta, Elisabeth; Elemento, Olivier; Melnick, Ari M.

    2012-01-01

    Genetic lesions such as BCR-ABL1, E2A-PBX1 and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point towards disease mechanisms and useful biomarkers and therapeutic targets. We therefore performed DNA methylation and gene expression profiling on a cohort of 215 adult B-ALL patients enrolled in a single phase III clinical trial (ECOG E2993) and normal control B-cells. In BCR-ABL1-positive B-ALL, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in ALL patients regardless of BCR-ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALL. In E2A-PBX1-positive B-ALL, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A-PBX1 ChIP sequencing (ChIP-seq), suggesting that E2A-PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6 targeted therapy as a new therapeutic strategy for MLLr B-ALL. PMID:23107779

  2. SPARQL-enabled identifier conversion with Identifiers.org

    PubMed Central

    Wimalaratne, Sarala M.; Bolleman, Jerven; Juty, Nick; Katayama, Toshiaki; Dumontier, Michel; Redaschi, Nicole; Le Novère, Nicolas; Hermjakob, Henning; Laibe, Camille

    2015-01-01

    Motivation: On the semantic web, in life sciences in particular, data is often distributed via multiple resources. Each of these sources is likely to use their own International Resource Identifier for conceptually the same resource or database record. The lack of correspondence between identifiers introduces a barrier when executing federated SPARQL queries across life science data. Results: We introduce a novel SPARQL-based service to enable on-the-fly integration of life science data. This service uses the identifier patterns defined in the Identifiers.org Registry to generate a plurality of identifier variants, which can then be used to match source identifiers with target identifiers. We demonstrate the utility of this identifier integration approach by answering queries across major producers of life science Linked Data. Availability and implementation: The SPARQL-based identifier conversion service is available without restriction at http://identifiers.org/services/sparql. Contact: sarala@ebi.ac.uk PMID:25638809

  3. SPARQL-enabled identifier conversion with Identifiers.org.

    PubMed

    Wimalaratne, Sarala M; Bolleman, Jerven; Juty, Nick; Katayama, Toshiaki; Dumontier, Michel; Redaschi, Nicole; Le Novère, Nicolas; Hermjakob, Henning; Laibe, Camille

    2015-06-01

    On the semantic web, in life sciences in particular, data is often distributed via multiple resources. Each of these sources is likely to use their own International Resource Identifier for conceptually the same resource or database record. The lack of correspondence between identifiers introduces a barrier when executing federated SPARQL queries across life science data. We introduce a novel SPARQL-based service to enable on-the-fly integration of life science data. This service uses the identifier patterns defined in the Identifiers.org Registry to generate a plurality of identifier variants, which can then be used to match source identifiers with target identifiers. We demonstrate the utility of this identifier integration approach by answering queries across major producers of life science Linked Data. The SPARQL-based identifier conversion service is available without restriction at http://identifiers.org/services/sparql. © The Author 2015. Published by Oxford University Press.

  4. Targeted delivery of non-viral vectors to cartilage in vivo using a chondrocyte-homing peptide identified by phage display.

    PubMed

    Pi, Yanbin; Zhang, Xin; Shi, Junjun; Zhu, Jinxian; Chen, Wenqing; Zhang, Chenguang; Gao, Weiwei; Zhou, Chunyan; Ao, Yingfang

    2011-09-01

    Gene therapy is a promising method for osteoarthritis and cartilage injury. However, specifically delivering target genes into chondrocytes is a great challenge because of their non-vascularity and the dense extracellular matrix of cartilage. In our study, we identified a chondrocyte-affinity peptide (CAP, DWRVIIPPRPSA) by phage display technology. Subsequent analysis suggests that the peptide can efficiently interact specifically with chondrocytes without any species specificity. Polyethylenimine (PEI) was covalently modified with CAP to construct a non-viral vector for cartilage-targeted therapy. To investigate the cartilage-targeting property of the CAP-modified vector, FITC-labeled CAP conjugated PEI/DNA particles were injected into rabbit knee joints, and visualized under confocal microscope. Higher concentrations of CAP-modified vector were detected in the cartilage and specifically taken up by chondrocytes compared with a randomly scrambled peptide (SP)-modified vector. To evaluate cartilage-targeting transfection efficiency, the GFP and luciferase genes were delivered into knee joints using CAP- and SP-modified PEI. Cartilage transfections mediated by CAP-modified PEI were much more efficient and specific than those by SP-modified PEI. This result suggests that CAP-modified PEI could be used as a specific cartilage-targeting vector for cartilage disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Genetic Variants Identified from Epilepsy of Unknown Etiology in Chinese Children by Targeted Exome Sequencing

    PubMed Central

    Wang, Yimin; Du, Xiaonan; Bin, Rao; Yu, Shanshan; Xia, Zhezhi; Zheng, Guo; Zhong, Jianmin; Zhang, Yunjian; Jiang, Yong-hui; Wang, Yi

    2017-01-01

    Genetic factors play a major role in the etiology of epilepsy disorders. Recent genomics studies using next generation sequencing (NGS) technique have identified a large number of genetic variants including copy number (CNV) and single nucleotide variant (SNV) in a small set of genes from individuals with epilepsy. These discoveries have contributed significantly to evaluate the etiology of epilepsy in clinic and lay the foundation to develop molecular specific treatment. However, the molecular basis for a majority of epilepsy patients remains elusive, and furthermore, most of these studies have been conducted in Caucasian children. Here we conducted a targeted exome-sequencing of 63 trios of Chinese epilepsy families using a custom-designed NGS panel that covers 412 known and candidate genes for epilepsy. We identified pathogenic and likely pathogenic variants in 15 of 63 (23.8%) families in known epilepsy genes including SCN1A, CDKL5, STXBP1, CHD2, SCN3A, SCN9A, TSC2, MBD5, POLG and EFHC1. More importantly, we identified likely pathologic variants in several novel candidate genes such as GABRE, MYH1, and CLCN6. Our results provide the evidence supporting the application of custom-designed NGS panel in clinic and indicate a conserved genetic susceptibility for epilepsy between Chinese and Caucasian children. PMID:28074849

  6. Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

    PubMed Central

    Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

    2013-01-01

    Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

  7. Salivary Myeloperoxidase, Assessed by 3,3′-Diaminobenzidine Colorimetry, Can Differentiate Periodontal Patients from Nonperiodontal Subjects

    PubMed Central

    Klangprapan, Supaporn; Chaiyarit, Ponlatham; Hormdee, Doosadee; Kampichai, Amonrujee; Khampitak, Tueanjit; Daduang, Jureerut; Tavichakorntrakool, Ratree; Panijpan, Bhinyo; Boonsiri, Patcharee

    2016-01-01

    Periodontal diseases, which result from inflammation of tooth supporting tissues, are highly prevalent worldwide. Myeloperoxidase (MPO), from certain white blood cells in saliva, is a biomarker for inflammation. We report our study on the salivary MPO activity and its association with severity of periodontal diseases among Thai patients. Periodontally healthy subjects (n = 11) and gingivitis (n = 32) and periodontitis patients (n = 19) were enrolled. Assessments of clinically periodontal parameters were reported as percentages for gingival bleeding index (GI) and bleeding on probing (BOP), whereas pocket depth (PD) and clinical attachment loss (CAL) were measured in millimeters and then made to index scores. Salivary MPO activity was measured by colorimetry using 3,3′-diaminobenzidine as substrate. The results showed that salivary MPO activity in periodontitis patients was significantly higher than in healthy subjects (p = 0.003) and higher than in gingivitis patients (p = 0.059). No difference was found between gingivitis and healthy groups (p = 0.181). Significant correlations were observed (p < 0.01) between salivary MPO activity and GI (r = 0.632, p < 0.001), BOP (r = 0.599, p < 0.001), PD (r = 0.179, p = 0.164), and CAL (r = 0.357, p = 0.004) index scores. Sensitivity (94.12%), specificity (54.55%), and positive (90.57%) and negative (66.67%) predictive values indicate that salivary MPO activity has potential use as a screening marker for oral health of the Thai community. PMID:27274868

  8. Drug synergy screen and network modeling in dedifferentiated liposarcoma identifies CDK4 and IGF1R as synergistic drug targets.

    PubMed

    Miller, Martin L; Molinelli, Evan J; Nair, Jayasree S; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M; Singer, Samuel; Schwartz, Gary K; Sander, Chris

    2013-09-24

    Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depends on the activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.

  9. A qualitative study of GP, nurse and practice manager views on using targeted case-finding to identify patients with COPD in primary care.

    PubMed

    Summers, Rachael H; Sharmeen, Taniya; Lippiett, Kate; Gillett, Kate; Astles, Carla; Vu, Linh; Stafford-Watson, Mark; Bruton, Anne; Thomas, Mike; Wilkinson, Tom

    2017-08-29

    'Finding the missing millions' with chronic obstructive pulmonary disease became part of the Department of Health strategy for England in 2010. Targeted case-finding within primary care is one potential pro-active strategy, but currently little is known about the views of healthcare professionals on this approach. In this study, 36 healthcare professionals (12 GPs, 14 nurses, and 10 practice managers) from 34 UK practices participated in semi-structured telephone interviews about targeted case-finding. Interviews followed an interview guide, were audio-recorded, transcribed verbatim, coded and analysed using 'Framework Approach'. Most of those interviewed practiced opportunistic case-finding. The main perceived barriers to wider case-finding programmes were the resource implications associated with running such programmes and identifying more chronic obstructive pulmonary disease patients. Financial incentives, support from specialist clinicians, and comprehensive guidance were viewed as facilitators. While targeted case-finding is conceptually accepted by primary care staff, scepticism surrounding (1) the value of identifying those with mild disease and (2) the availability of effective targeted case-finding methods, may lead some to favour an opportunistic approach. Key concerns were a lack of unequivocal evidence for the relative benefits vs. disadvantages of diagnosing patients earlier, and resource constraints in an already over-burdened system. Barriers to practical implementation of case-finding studies may be addressed with financial, human and educational resources, such as additional staff to undertake searches and perform spirometry tests, and practical and educational support from specialist teams. SUPPORT NEEDED TO IDENTIFY THOSE UNDIAGNOSED: Additional staff and resources would facilitate targeted searches for patients showing symptoms of early-stage chronic lung disease. Chronic obstructive pulmonary disease (COPD) costs the UK economy billions of

  10. Three-step HPLC-ESI-MS/MS procedure for screening and identifying non-target flavonoid derivatives

    NASA Astrophysics Data System (ADS)

    Rak, Gábor; Fodor, Péter; Abrankó, László

    2010-02-01

    A three-step HPLC-ESI-MS/MS procedure is designed for screening and identification of non-target flavonoid derivatives of selected flavonoid aglycones. In this method the five commonly appearing aglycones (apigenin, luteolin, myricetin, naringenin and quercetin) were selected. The method consists of three individual mass spectrometric experiments of which the first two were implemented within a single chromatographic acquisition. The third step was carried out during a replicate chromatographic run using the same RP-HPLC conditions. The first step, a multiple reaction monitoring (MRM) scan of the aglycones was performed to define the number of derivatives relating to the selected aglycones. For this purpose the characteristic aglycone parts of the unknowns were used as specific tags of the molecules, which were generated as in-source fragments. Secondly, a full scan MS experiment is performed to identify the masses of the potential derivatives of the selected aglycones. Finally, the third step had the capability to confirm the supposed derivatives. The developed method was applied to a commercially available black currant juice to demonstrate its capability to detect and identify various flavonoid glycosides without any preliminary information about their presence in the sample. As a result 13 compounds were detected and identified in total. Namely, 3 different myricetin glycosides and the myricetin aglycone 2 luteolin glycosides plus the aglycone and 3 quercetin glycosides plus the aglycone could be identified from the tested black currant sample. In the case of apigenin and naringenin only the aglycones could be detected.

  11. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sarró, Eduard, E-mail: eduard.sarro@vhir.org; Renal Physiopathology, CIBBIM-Nanomedicine, Vall d'Hebron Research Institute; Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC.more » Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also

  12. Evaluation of the serum catalase and myeloperoxidase activities in chronic arsenic-exposed individuals and concomitant cytogenetic damage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Banerjee, Mayukh; Banerjee, Nilanjana; Ghosh, Pritha

    2010-11-15

    Chronic arsenic exposure through contaminated drinking water is a major environmental health issue. Chronic arsenic exposure is known to exert its toxic effects by a variety of mechanisms, of which generation of reactive oxygen species (ROS) is one of the most important. A high level of ROS, in turn, leads to DNA damage that might ultimately culminate in cancer. In order to keep the level of ROS in balance, an array of enzymes is present, of which catalase (CAT) and myeloperoxidase (MPO) are important members. Hence, in this study, we determined the activities of these two enzymes in the seramore » and chromosomal aberrations (CA) in peripheral blood lymphocytes in individuals exposed and unexposed to arsenic in drinking water. Arsenic in drinking water and in urine was used as a measure of exposure. Our results show that individuals chronically exposed to arsenic have significantly higher CAT and MPO activities and higher incidence of CA. We found moderate positive correlations between CAT and MPO activities, induction of CA and arsenic in urine and water. These results indicate that chronic arsenic exposure causes higher CAT and MPO activities in serum that correlates with induction of genetic damage. We conclude that the serum levels of these enzymes might be used as biomarkers of early arsenic exposure induced disease much before the classical dermatological symptoms of arsenicosis begin to appear.« less

  13. An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased expression of the homeostatic regulator pumilio.

    PubMed

    Lin, Wei-Hsiang; He, Miaomiao; Fan, Yuen Ngan; Baines, Richard A

    2018-05-02

    Despite availability of a diverse range of anti-epileptic drugs (AEDs), only about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel targets are required to catalyse the design of next-generation AEDs. Manipulation of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has been shown to be potently anticonvulsant in Drosophila. In this study, we performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum activity reporter and identified 1166 genes involved in dPum regulation. Of these genes, we focused on 699 genes that, on knock-down, potentiate dPum activity/expression. Of this subgroup, 101 genes are activity-dependent based on comparison with genes previously identified as activity-dependent by RNA-sequencing. Functional cluster analysis shows these genes are enriched in pathways involved in DNA damage, regulation of cell cycle and proteasomal protein catabolism. To test for anticonvulsant activity, we utilised an RNA-interference approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are sufficient to significantly reduce seizure duration in the characterized seizure mutant, para bss . We further show that chemical inhibitors of protein products of some of the genes targeted are similarly anticonvulsant. Finally, to establish whether the anticonvulsant activity of identified compounds results from increased dpum transcription, we performed a luciferase-based assay to monitor dpum promoter activity. Third instar larvae exposed to sodium fluoride, gemcitabine, metformin, bestatin, WP1066 or valproic acid all showed increased dpum promoter activity. Thus, this study validates Pum as a favourable target for AED design and, moreover, identifies a number of lead compounds capable of increasing the expression of this homeostatic regulator.

  14. OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

    PubMed Central

    Ranganathan, Sridhar; Suthers, Patrick F.; Maranas, Costas D.

    2010-01-01

    Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis. PMID:20419153

  15. A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35–NP Interface

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Gai; Nash, Peter J.; Johnson, Britney

    The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarizationmore » assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP–VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.« less

  16. A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

    PubMed

    Saso, Wakana; Tsukuda, Senko; Ohashi, Hirofumi; Fukano, Kento; Morishita, Ryo; Matsunaga, Satoko; Ohki, Mio; Ryo, Akihide; Park, Sam-Yong; Suzuki, Ryosuke; Aizaki, Hideki; Muramatsu, Masamichi; Sureau, Camille; Wakita, Takaji; Matano, Tetsuro; Watashi, Koichi

    2018-06-22

    Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Emory University: MEDICI (Mining Essentiality Data to Identify Critical Interactions) for Cancer Drug Target Discovery and Development | Office of Cancer Genomics

    Cancer.gov

    The CTD2 Center at Emory University has developed a computational methodology to combine high-throughput knockdown data with known protein network topologies to infer the importance of protein-protein interactions (PPIs) for the survival of cancer cells.  Applying these data to the Achilles shRNA results, the CCLE cell line characterizations, and known and newly identified PPIs provides novel insights for potential new drug targets for cancer therapies and identifies important PPI hubs.

  18. Wide screening of phage-displayed libraries identifies immune targets in planta.

    PubMed

    Rioja, Cristina; Van Wees, Saskia C; Charlton, Keith A; Pieterse, Corné M J; Lorenzo, Oscar; García-Sánchez, Susana

    2013-01-01

    Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well

  19. Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

    PubMed

    Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

    2012-01-01

    HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

  20. Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

    PubMed Central

    Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

    2012-01-01

    HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

  1. Identifying Changes in Youth's Subgroup Membership over Time Based on Their Targeted Communication about Substance Use with Parents and Friends

    ERIC Educational Resources Information Center

    Kam, Jennifer A.

    2011-01-01

    Using latent class/transition analyses, this study: (a) identified subgroups of youth based on their targeted communication about substance use with parents and friends, (b) examined subgroup differences in substance use, and (c) considered changes in subgroup membership over four years. Among 5,874 youth, five subgroups emerged, with parents-only…

  2. An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors including DNA repair proteins and Nrf2

    PubMed Central

    Cardnell, Robert J.G.; Behrens, Carmen; Diao, Lixia; Fan, YouHong; Tang, Ximing; Tong, Pan; John D., Minna; Mills, Gordon B.; Heymach, John V.; Wistuba, Ignacio I.; Wang, Jing; Byers., Lauren A.

    2015-01-01

    Purpose Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15-20%) does not express TTF1 and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets. Experimental Design Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and the TCGA, we interrogated proteomic profiles (by reverse-phase protein array (RPPA)), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents. Results Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho=0.57, p<0.001) and gene expression (NKX2-1, Rho=0.61, p<0.001). Established driver mutations (e.g. BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (p=0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3K/MAPK signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines. Conclusions Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD includingKEAP1/Nrf2 and DNA repair pathways. PMID:25878335

  3. Drug Synergy Screen and Network Modeling in Dedifferentiated Liposarcoma Identifies CDK4 and IGF1R as Synergistic Drug Targets

    PubMed Central

    Miller, Martin L.; Molinelli, Evan J.; Nair, Jayasree S.; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M.; Singer, Samuel; Schwartz, Gary K.; Sander, Chris

    2014-01-01

    Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depend on activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies. PMID:24065146

  4. Identifying Molecular Targets for Chemoprevention in a Rat Model

    DTIC Science & Technology

    2005-12-01

    95616-8671 REPORT DATE: December 2005 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command...California 95616-8671 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and...addition, it induces high levels of oxidative damage in the target tissues. The scope of this research includes: 1) Generation of a rat model, 2) Analysis

  5. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

    PubMed Central

    Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R.; Herrmann, Harald; Sison, Edward A.; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J.; Johns, Christopher; Chicas, Agustin; Mulloy, James C.; Kogan, Scott C.; Brown, Patrick; Valent, Peter; Bradner, James E.; Lowe, Scott W.; Vakoc, Christopher R.

    2012-01-01

    Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention. PMID:21814200

  6. The C3aR promotes macrophage infiltration and regulates ANCA production but does not affect glomerular injury in experimental anti-myeloperoxidase glomerulonephritis

    PubMed Central

    Gan, Poh-Yi; Kitching, A. Richard; Holdsworth, Stephen R.

    2018-01-01

    The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides are autoimmune diseases associated with significant morbidity and mortality. They often affect the kidney causing rapidly progressive glomerulonephritis. While signalling by complement anaphylatoxin C5a though the C5a receptor is important in this disease, the role of the anaphylatoxin C3a signalling via the C3a receptor (C3aR) is not known. Using two different murine models of anti-myeloperoxidase (MPO) glomerulonephritis, one mediated by passive transfer of anti-MPO antibodies, the other by cell-mediated immunity, we found that the C3aR did not alter histological disease severity. However, it promoted macrophage recruitment to the inflamed glomerulus and inhibited the generation of MPO-ANCA whilst not influencing T cell autoimmunity. Thus, whilst the C3aR modulates some elements of disease pathogenesis, overall it is not critical in effector responses and glomerular injury caused by autoimmunity to MPO. PMID:29315316

  7. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets

    PubMed Central

    Vinayagam, Arunachalam; Gibson, Travis E.; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-01-01

    The protein–protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as “indispensable,” “neutral,” or “dispensable,” which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network’s control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

  8. Targeted DNA sequencing of non-small cell lung cancer identifies mutations associated with brain metastases.

    PubMed

    Wilson, George D; Johnson, Matthew D; Ahmed, Samreen; Cardenas, Paola Yumpo; Grills, Inga S; Thibodeau, Bryan J

    2018-05-25

    This study explores the hypothesis that dominant molecular oncogenes in non-small cell lung cancer (NSCLC) are associated with metastatic spread to the brain. NSCLC patient groups with no evidence of metastasis, with metastatic disease to a non-CNS site, who developed brain metastasis after diagnosis, and patients with simultaneous diagnosis of NSCLC and metastatic brain lesions were studied using targeted sequencing. In patients with brain metastasis versus those without, only 2 variants (one each in BCL6 and NOTHC2) were identified that occurred in ≥ 4 NSCLC of patients with brain metastases but ≤ 1 of the NSCLC samples without brain metastases. At the gene level, 20 genes were found to have unique variants in more than 33% of the patients with brain metastases. When analyzed at the patient level, these 20 genes formed the basis of a predictive test to discriminate those with brain metastasis. Further analysis showed that PI3K/AKT signaling is altered in both the primary and metastases of NSCLC patients with brain lesions. While no single variant was associated with brain metastasis, this study describes a potential gene panel for the identification of patients at risk and implicates PI3K/AKT signaling as a therapeutic target.

  9. Targeted DNA sequencing of non-small cell lung cancer identifies mutations associated with brain metastases

    PubMed Central

    Wilson, George D.; Johnson, Matthew D.; Ahmed, Samreen; Cardenas, Paola Yumpo; Grills, Inga S.; Thibodeau, Bryan J.

    2018-01-01

    Introduction This study explores the hypothesis that dominant molecular oncogenes in non-small cell lung cancer (NSCLC) are associated with metastatic spread to the brain. Methods NSCLC patient groups with no evidence of metastasis, with metastatic disease to a non-CNS site, who developed brain metastasis after diagnosis, and patients with simultaneous diagnosis of NSCLC and metastatic brain lesions were studied using targeted sequencing. Results In patients with brain metastasis versus those without, only 2 variants (one each in BCL6 and NOTHC2) were identified that occurred in ≥ 4 NSCLC of patients with brain metastases but ≤ 1 of the NSCLC samples without brain metastases. At the gene level, 20 genes were found to have unique variants in more than 33% of the patients with brain metastases. When analyzed at the patient level, these 20 genes formed the basis of a predictive test to discriminate those with brain metastasis. Further analysis showed that PI3K/AKT signaling is altered in both the primary and metastases of NSCLC patients with brain lesions. Conclusion While no single variant was associated with brain metastasis, this study describes a potential gene panel for the identification of patients at risk and implicates PI3K/AKT signaling as a therapeutic target. PMID:29899834

  10. The Human Kinome Targeted by FDA Approved Multi-Target Drugs and Combination Products: A Comparative Study from the Drug-Target Interaction Network Perspective.

    PubMed

    Li, Ying Hong; Wang, Pan Pan; Li, Xiao Xu; Yu, Chun Yan; Yang, Hong; Zhou, Jin; Xue, Wei Wei; Tan, Jun; Zhu, Feng

    2016-01-01

    The human kinome is one of the most productive classes of drug target, and there is emerging necessity for treating complex diseases by means of polypharmacology (multi-target drugs and combination products). However, the advantages of the multi-target drugs and the combination products are still under debate. A comparative analysis between FDA approved multi-target drugs and combination products, targeting the human kinome, was conducted by mapping targets onto the phylogenetic tree of the human kinome. The approach of network medicine illustrating the drug-target interactions was applied to identify popular targets of multi-target drugs and combination products. As identified, the multi-target drugs tended to inhibit target pairs in the human kinome, especially the receptor tyrosine kinase family, while the combination products were able to against targets of distant homology relationship. This finding asked for choosing the combination products as a better solution for designing drugs aiming at targets of distant homology relationship. Moreover, sub-networks of drug-target interactions in specific disease were generated, and mechanisms shared by multi-target drugs and combination products were identified. In conclusion, this study performed an analysis between approved multi-target drugs and combination products against the human kinome, which could assist the discovery of next generation polypharmacology.

  11. Applying the Theoretical Domains Framework to identify barriers and targeted interventions to enhance nurses' use of electronic medication management systems in two Australian hospitals.

    PubMed

    Debono, Deborah; Taylor, Natalie; Lipworth, Wendy; Greenfield, David; Travaglia, Joanne; Black, Deborah; Braithwaite, Jeffrey

    2017-03-27

    Medication errors harm hospitalised patients and increase health care costs. Electronic Medication Management Systems (EMMS) have been shown to reduce medication errors. However, nurses do not always use EMMS as intended, largely because implementation of such patient safety strategies requires clinicians to change their existing practices, routines and behaviour. This study uses the Theoretical Domains Framework (TDF) to identify barriers and targeted interventions to enhance nurses' appropriate use of EMMS in two Australian hospitals. This qualitative study draws on in-depth interviews with 19 acute care nurses who used EMMS. A convenience sampling approach was used. Nurses working on the study units (N = 6) in two hospitals were invited to participate if available during the data collection period. Interviews inductively explored nurses' experiences of using EMMS (step 1). Data were analysed using the TDF to identify theory-derived barriers to nurses' appropriate use of EMMS (step 2). Relevant behaviour change techniques (BCTs) were identified to overcome key barriers to using EMMS (step 3) followed by the identification of potential literature-informed targeted intervention strategies to operationalise the identified BCTs (step 4). Barriers to nurses' use of EMMS in acute care were represented by nine domains of the TDF. Two closely linked domains emerged as major barriers to EMMS use: Environmental Context and Resources (availability and properties of computers on wheels (COWs); technology characteristics; specific contexts; competing demands and time pressure) and Social/Professional Role and Identity (conflict between using EMMS appropriately and executing behaviours critical to nurses' professional role and identity). The study identified three potential BCTs to address the Environmental Context and Resources domain barrier: adding objects to the environment; restructuring the physical environment; and prompts and cues. Seven BCTs to address Social

  12. Association of Major Depressive Disorder with Serum Myeloperoxidase and other Markers of Inflammation: A Twin Study

    PubMed Central

    Vaccarino, Viola; Brennan, Marie-Luise; Miller, Andrew H.; Bremner, J. Douglas; Ritchie, James C.; Lindau, Frauke; Veledar, Emir; Su, Shaoyong; Murrah, Nancy V.; Jones, Linda; Jawed, Farhan; Dai, Jun; Goldberg, Jack; Hazen, Stanley L.

    2008-01-01

    Background Major depressive disorder (MDD) has been linked to inflammation, but this association may be due to common precursors to both depression and inflammation. Myeloperoxidase (MPO) is an inflammatory enzyme produced by activated leukocytes which predicts risk of coronary heart disease. We sought to examine whether MPO and other markers of inflammation are associated with MDD, and whether the association is confounded by genetic or other shared familial factors. Methods We examined 178 monozygotic and dizygotic middle-aged male twin pairs. We assessed MDD with the Structured Clinical Interview for Psychiatry Disorders. Blood markers of inflammation included MPO, interleukin-6, white blood cell count, C-reactive protein, tumor necrosis factor (TNF)-α, the TNF-α soluble receptor II, and fibrinogen. Analyses were conducted in the overall sample and among 67 twin pairs discordant for MDD using mixed effects regression. Results Twins with a history of MDD had 32% higher levels of MPO (p<0.0001); this difference persisted after adjusting for other risk factors. Among dizygotic MDD-discordant twin pairs, twins with MDD had 77% higher MPO than their brothers without MDD after adjusting for other factors (p<0.0001). In contrast, no significant association was found in monozygotic twins (p=0.13). Similar, but weaker, associations were found between MDD and other inflammatory biomarkers. Conclusion MPO is a useful biomarker of immune activation in MDD. However, the association between inflammation and MDD is largely due to common genetic liability. Our results are consistent with the hypothesis that genes promoting inflammation are involved in the pathogenesis of MDD. PMID:18514165

  13. Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

    PubMed

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-03-11

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Comprehensive Analyses of Ventricular Myocyte Models Identify Targets Exhibiting Favorable Rate Dependence

    PubMed Central

    Bugana, Marco; Severi, Stefano; Sobie, Eric A.

    2014-01-01

    Reverse rate dependence is a problematic property of antiarrhythmic drugs that prolong the cardiac action potential (AP). The prolongation caused by reverse rate dependent agents is greater at slow heart rates, resulting in both reduced arrhythmia suppression at fast rates and increased arrhythmia risk at slow rates. The opposite property, forward rate dependence, would theoretically overcome these parallel problems, yet forward rate dependent (FRD) antiarrhythmics remain elusive. Moreover, there is evidence that reverse rate dependence is an intrinsic property of perturbations to the AP. We have addressed the possibility of forward rate dependence by performing a comprehensive analysis of 13 ventricular myocyte models. By simulating populations of myocytes with varying properties and analyzing population results statistically, we simultaneously predicted the rate-dependent effects of changes in multiple model parameters. An average of 40 parameters were tested in each model, and effects on AP duration were assessed at slow (0.2 Hz) and fast (2 Hz) rates. The analysis identified a variety of FRD ionic current perturbations and generated specific predictions regarding their mechanisms. For instance, an increase in L-type calcium current is FRD when this is accompanied by indirect, rate-dependent changes in slow delayed rectifier potassium current. A comparison of predictions across models identified inward rectifier potassium current and the sodium-potassium pump as the two targets most likely to produce FRD AP prolongation. Finally, a statistical analysis of results from the 13 models demonstrated that models displaying minimal rate-dependent changes in AP shape have little capacity for FRD perturbations, whereas models with large shape changes have considerable FRD potential. This can explain differences between species and between ventricular cell types. Overall, this study provides new insights, both specific and general, into the determinants of AP duration

  15. Comprehensive analyses of ventricular myocyte models identify targets exhibiting favorable rate dependence.

    PubMed

    Cummins, Megan A; Dalal, Pavan J; Bugana, Marco; Severi, Stefano; Sobie, Eric A

    2014-03-01

    Reverse rate dependence is a problematic property of antiarrhythmic drugs that prolong the cardiac action potential (AP). The prolongation caused by reverse rate dependent agents is greater at slow heart rates, resulting in both reduced arrhythmia suppression at fast rates and increased arrhythmia risk at slow rates. The opposite property, forward rate dependence, would theoretically overcome these parallel problems, yet forward rate dependent (FRD) antiarrhythmics remain elusive. Moreover, there is evidence that reverse rate dependence is an intrinsic property of perturbations to the AP. We have addressed the possibility of forward rate dependence by performing a comprehensive analysis of 13 ventricular myocyte models. By simulating populations of myocytes with varying properties and analyzing population results statistically, we simultaneously predicted the rate-dependent effects of changes in multiple model parameters. An average of 40 parameters were tested in each model, and effects on AP duration were assessed at slow (0.2 Hz) and fast (2 Hz) rates. The analysis identified a variety of FRD ionic current perturbations and generated specific predictions regarding their mechanisms. For instance, an increase in L-type calcium current is FRD when this is accompanied by indirect, rate-dependent changes in slow delayed rectifier potassium current. A comparison of predictions across models identified inward rectifier potassium current and the sodium-potassium pump as the two targets most likely to produce FRD AP prolongation. Finally, a statistical analysis of results from the 13 models demonstrated that models displaying minimal rate-dependent changes in AP shape have little capacity for FRD perturbations, whereas models with large shape changes have considerable FRD potential. This can explain differences between species and between ventricular cell types. Overall, this study provides new insights, both specific and general, into the determinants of AP duration

  16. Melatonin Prevents Myeloperoxidase Heme Destruction and the Generation of Free Iron Mediated by Self-Generated Hypochlorous Acid

    PubMed Central

    Shaeib, Faten; Khan, Sana N.; Ali, Iyad; Najafi, Tohid; Maitra, Dhiman; Abdulhamid, Ibrahim; Saed, Ghassan M.; Pennathur, Subramaniam; Abu-Soud, Husam M.

    2015-01-01

    Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl–mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation. PMID:25835505

  17. A multi-element screening method to identify metal targets for blood biomonitoring in green sea turtles (Chelonia mydas).

    PubMed

    Villa, C A; Finlayson, S; Limpus, C; Gaus, C

    2015-04-15

    Biomonitoring of blood is commonly used to identify and quantify occupational or environmental exposure to chemical contaminants. Increasingly, this technique has been applied to wildlife contaminant monitoring, including for green turtles, allowing for the non-lethal evaluation of chemical exposure in their nearshore environment. The sources, composition, bioavailability and toxicity of metals in the marine environment are, however, often unknown and influenced by numerous biotic and abiotic factors. These factors can vary considerably across time and space making the selection of the most informative elements for biomonitoring challenging. This study aimed to validate an ICP-MS multi-element screening method for green turtle blood in order to identify and facilitate prioritisation of target metals for subsequent fully quantitative analysis. Multi-element screening provided semiquantitative results for 70 elements, 28 of which were also determined through fully quantitative analysis. Of the 28 comparable elements, 23 of the semiquantitative results had an accuracy between 67% and 112% relative to the fully quantified values. In lieu of any available turtle certified reference materials (CRMs), we evaluated the use of human blood CRMs as a matrix surrogate for quality control, and compared two commonly used sample preparation methods for matrix related effects. The results demonstrate that human blood provides an appropriate matrix for use as a quality control material in the fully quantitative analysis of metals in turtle blood. An example for the application of this screening method is provided by comparing screening results from blood of green turtles foraging in an urban and rural region in Queensland, Australia. Potential targets for future metal biomonitoring in these regions were identified by this approach. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Ras-Driven Transcriptome Analysis Identifies Aurora Kinase A as a Potential Malignant Peripheral Nerve Sheath Tumor Therapeutic Target

    PubMed Central

    Patel, Ami V.; Eaves, David; Jessen, Walter J.; Rizvi, Tilat A.; Ecsedy, Jeffrey A.; Qian, Mark G.; Aronow, Bruce J.; Perentesis, John P.; Serra, Eduard; Cripe, Timothy P.; Miller, Shyra J.; Ratner, Nancy

    2013-01-01

    Purpose Patients with Neurofibromatosis Type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST) which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to utilize comprehensive gene expression analysis to identify novel therapeutic targets. Experimental Design Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST due to the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively-active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase over-expression in MPNST in vitro and in vivo using Aurora kinase shRNAs and compounds that inhibit Aurora kinase. Results We identified 2000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically over-expressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. Conclusion Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. PMID:22811580

  19. Identifying new lignin bioengineering targets: impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and fermentation of maize cell walls

    USDA-ARS?s Scientific Manuscript database

    Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, we artificially lignified maize cell walls with normal monolignols pl...

  20. Mechanisms of hypochlorite injury of target cells.

    PubMed Central

    Schraufstätter, I U; Browne, K; Harris, A; Hyslop, P A; Jackson, J H; Quehenberger, O; Cochrane, C G

    1990-01-01

    HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants. PMID:2153710

  1. Identifying vaccine targets for anti-leishmanial vaccine development

    PubMed Central

    Sundar, Shyam; Singh, Bhawana

    2014-01-01

    Leishmaniasis is a neglected tropical disease spread by an arthropod vector. It remains a significant health problem with an incidence of 0.2–0.4 million VL and 0.7–1.2 million CL cases each year. There are limitations associated with the current therapeutic regimens for leishmaniasis and the fact that after recovery from infection the host becomes immune to subsequent infection therefore, these factors forces the feasibility of a vaccine for leishmaniasis. Publication of the genome sequence of Leishmania has paved a new way to understand the pathogenesis and host immunological status therefore providing a deep insight in the field of vaccine research. This review is an effort to study the antigenic targets in Leishmania to develop anti-leishmanial vaccine. PMID:24606556

  2. Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing.

    PubMed

    Lee, Paul J; Yoo, Naomi S; Hagemann, Ian S; Pfeifer, John D; Cottrell, Catherine E; Abel, Haley J; Duncavage, Eric J

    2017-02-01

    Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. In vivo Labeling of Constellations of Functionally Identified Neurons for Targeted in vitro Recordings

    PubMed Central

    Lien, Anthony D.; Scanziani, Massimo

    2011-01-01

    Relating the functional properties of neurons in an intact organism with their cellular and synaptic characteristics is necessary for a mechanistic understanding of brain function. However, while the functional properties of cortical neurons (e.g., tuning to sensory stimuli) are necessarily determined in vivo, detailed cellular and synaptic analysis relies on in vitro techniques. Here we describe an approach that combines in vivo calcium imaging (for functional characterization) with photo-activation of fluorescent proteins (for neuron labeling), thereby allowing targeted in vitro recording of multiple neurons with known functional properties. We expressed photo-activatable GFP rendered non-diffusible through fusion with a histone protein (H2B–PAGFP) in the mouse visual cortex to rapidly photo-label constellations of neurons in vivo at cellular and sub-cellular resolution using two-photon excitation. This photo-labeling method was compatible with two-photon calcium imaging of neuronal responses to visual stimuli, allowing us to label constellations of neurons with specific functional properties. Photo-labeled neurons were easily identified in vitro in acute brain slices and could be targeted for whole-cell recording. We also demonstrate that in vitro and in vivo image stacks of the same photo-labeled neurons could be registered to one another, allowing the exact in vivo response properties of individual neurons recorded in vitro to be known. The ability to perform in vitro recordings from neurons with known functional properties opens up exciting new possibilities for dissecting the cellular, synaptic, and circuit mechanisms that underlie neuronal function in vivo. PMID:22144948

  4. Myeloperoxidase-oxidized high density lipoprotein impairs atherosclerotic plaque stability by inhibiting smooth muscle cell migration.

    PubMed

    Zhou, Boda; Zu, Lingyun; Chen, Yong; Zheng, Xilong; Wang, Yuhui; Pan, Bing; Dong, Min; Zhou, Enchen; Zhao, Mingming; Zhang, Youyi; Zheng, Lemin; Gao, Wei

    2017-01-10

    High density lipoprotein (HDL) has been proved to be a protective factor for coronary heart disease. Notably, HDL in atherosclerotic plaques can be nitrated (NO 2 -oxHDL) and chlorinated (Cl-oxHDL) by myeloperoxidase (MPO), likely compromising its cardiovascular protective effects. Here we determined the effects of NO 2 -oxHDL and Cl-oxHDL on SMC migration using wound healing and transwell assays, proliferation using MTT and BrdU assays, and apoptosis using Annexin-V assay in vitro, as well as on atherosclerotic plaque stability in vivo using a coratid artery collar implantation mice model. Our results showed that native HDL promoted SMC proliferation and migration, whereas NO 2 -oxHDL and Cl-oxHDL inhibited SMC migration and reduced capacity of stimulating SMC proliferation as well as migration, respectively. OxHDL had no significant influence on SMC apoptosis. In addition, we found that ERK1/2-phosphorylation was significantly lower when SMCs were incubated with NO 2 -oxHDL and Cl-oxHDL. Furthermore, transwell experiments showed that differences between native HDL, NO 2 -oxHDL and Cl-oxHDL was abolished after PD98059 (MAPK kinase inhibitor) treatment. In aortic SMCs from scavenger receptor BI (SR-BI) deficient mice, differences between migration of native HDL, NO 2 -oxHDL and Cl-oxHDL treated SMCs vanished, indicating SR-BI's possible role in HDL-associated SMC migration. Importantly, NO 2 -oxHDL and Cl-oxHDL induced neointima formation and reduced SMC positive staining cells in atherosclerotic plaque, resulting in elevated vulnerable index of atherosclerotic plaque. These findings implicate MPO-catalyzed oxidization of HDL may contribute to atherosclerotic plaque instability by inhibiting SMC proliferation and migration through MAPK-ERK pathway which was dependent on SR-BI.

  5. A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

    NASA Astrophysics Data System (ADS)

    Bhajun, Ricky; Guyon, Laurent; Pitaval, Amandine; Sulpice, Eric; Combe, Stéphanie; Obeid, Patricia; Haguet, Vincent; Ghorbel, Itebeddine; Lajaunie, Christian; Gidrol, Xavier

    2015-02-01

    MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues

  6. Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the CMV Status

    PubMed Central

    Pérez-Bercoff, Lena; Valentini, Davide; Gaseitsiwe, Simani; Mahdavifar, Shahnaz; Schutkowski, Mike; Poiret, Thomas; Pérez-Bercoff, Åsa; Ljungman, Per; Maeurer, Markus J.

    2014-01-01

    Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution. PMID:24740411

  7. Race-Specific Associations of Myeloperoxidase with Atherosclerosis in a Population-Based Sample: The Dallas Heart Study

    PubMed Central

    Chen, Lu; Rohatgi, Anand; Ayers, Colby R.; Das, Sandeep R.; Khera, Amit; Berry, Jarett D.; McGuire, Darren K.; de Lemos, James A.

    2011-01-01

    Objective Myeloperoxidase (MPO) is a leukocyte-derived enzyme that appears to be directly involved in atherosclerosis development. We evaluated the association of circulating MPO with coronary and aortic atherosclerosis in a large, multiethnic population. Methods and Results Plasma levels of MPO were measured in 3294 subjects participating in the Dallas Heart Study, a probability-based population sample. Coronary artery calcification (CAC) was measured by EBCT, and abdominal aorta plaque prevalence (AP) and burden (APB), as well as abdominal aorta wall thickness (AWT) were determined by MRI. Associations between MPO and atherosclerosis phenotypes were assessed in multivariable analyses adjusting for traditional atherosclerosis risk factors. MPO levels in the 4th compared with 1st quartile independently associated with prevalent AP (OR 1.41, 95% CI 1.08–1.84), APB (beta coefficient 0.23, p=0.02), and AWT (beta coefficient 0.04, p=0.03), but not with prevalent CAC (OR 0.84, 95% CI 0.61–1.17). MPO remained associated with aortic atherosclerosis phenotypes but not coronary calcification after adjustment for other inflammatory biomarkers. A significant interaction was observed between race/ethnicity, MPO and AP (pinteraction=0.038), such that MPO levels in the 4th vs 1st quartile associated with prevalent AP in African Americans, (OR 1.81, 95% CI 1.23–2.65) but not in White or Hispanic participants (OR 0.99, 95% CI 0.68–1.44). Conclusion Higher levels of MPO associated with aortic but not coronary atherosclerosis, with significant associations limited to African American participants. These findings suggest that MPO might be a novel risk factor contributing to racial disparities in peripheral vascular disease. PMID:21917261

  8. Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

    PubMed Central

    Victor, Victor M.; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Martinez de Marañon, Arantxa; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernández-Mijares, Antonio

    2016-01-01

    Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR. PMID:27007571

  9. Literature evidence in open targets - a target validation platform.

    PubMed

    Kafkas, Şenay; Dunham, Ian; McEntyre, Johanna

    2017-06-06

    We present the Europe PMC literature component of Open Targets - a target validation platform that integrates various evidence to aid drug target identification and validation. The component identifies target-disease associations in documents and ranks the documents based on their confidence from the Europe PMC literature database, by using rules utilising expert-provided heuristic information. The confidence score of a given document represents how valuable the document is in the scope of target validation for a given target-disease association by taking into account the credibility of the association based on the properties of the text. The component serves the platform regularly with the up-to-date data since December, 2015. Currently, there are a total number of 1168365 distinct target-disease associations text mined from >26 million PubMed abstracts and >1.2 million Open Access full text articles. Our comparative analyses on the current available evidence data in the platform revealed that 850179 of these associations are exclusively identified by literature mining. This component helps the platform's users by providing the most relevant literature hits for a given target and disease. The text mining evidence along with the other types of evidence can be explored visually through https://www.targetvalidation.org and all the evidence data is available for download in json format from https://www.targetvalidation.org/downloads/data .

  10. Determining Epigenetic Targets: A Beginner's Guide to Identifying Genome Functionality Through Database Analysis.

    PubMed

    Hay, Elizabeth A; Cowie, Philip; MacKenzie, Alasdair

    2017-01-01

    There can now be little doubt that the cis-regulatory genome represents the largest information source within the human genome essential for health. In addition to containing up to five times more information than the coding genome, the cis-regulatory genome also acts as a major reservoir of disease-associated polymorphic variation. The cis-regulatory genome, which is comprised of enhancers, silencers, promoters, and insulators, also acts as a major functional target for epigenetic modification including DNA methylation and chromatin modifications. These epigenetic modifications impact the ability of cis-regulatory sequences to maintain tissue-specific and inducible expression of genes that preserve health. There has been limited ability to identify and characterize the functional components of this huge and largely misunderstood part of the human genome that, for decades, was ignored as "Junk" DNA. In an attempt to address this deficit, the current chapter will first describe methods of identifying and characterizing functional elements of the cis-regulatory genome at a genome-wide level using databases such as ENCODE, the UCSC browser, and NCBI. We will then explore the databases on the UCSC genome browser, which provides access to DNA methylation and chromatin modification datasets. Finally, we will describe how we can superimpose the huge volume of study data contained in the NCBI archives onto that contained within the UCSC browser in order to glean relevant in vivo study data for any locus within the genome. An ability to access and utilize these information sources will become essential to informing the future design of experiments and subsequent determination of the role of epigenetics in health and disease and will form a critical step in our development of personalized medicine.

  11. Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

    PubMed

    Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

    2014-04-16

    The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

  12. Evaluation of usefulness of myeloperoxidase index (MPXI) for differential diagnosis of systemic inflammatory response syndrome (SIRS) in the emergency department.

    PubMed

    Cha, Yong Sung; Yoon, Jeong Min; Jung, Woo Jin; Kim, Yong Won; Kim, Tae Hoon; Kim, Oh Hyun; Cha, Kyoung Chul; Kim, Hyun; Hwang, Sung Oh; Lee, Kang Hyun

    2015-04-01

    The myeloperoxidase index (MPXI) is elevated in infection. We ascertained whether MPXI might be useful in differentiation of sepsis versus non-infectious systemic inflammatory response syndrome (SIRS) in emergency department (ED). After exclusion of patients with an age of <18 years, trauma, haematological disease and on anticancer chemotherapy, 444 consecutive cases with SIRS (sepsis: 224, 50.3%; and non-infectious SIRS: 220, 49.7%) diagnosed and treated at the ED of The Wonju Severance Christian Hospital from May 2012 to June 2012 were retrospectively reviewed. Median MPXI was higher in sepsis versus non-infectious SIRS (0.1 (IQR: -3.1 to 2.5) vs -1.2 (-4.1 to 1.6), respectively, p=0.020). Median white cell count, neutrophil percentage, C reactive protein level and δ neutrophil index were also higher. However, MPXI resulted as not statistically useful for differential diagnostic parameter in analysis. MPXI is higher in sepsis than in non-infectious SIRS. However, there is currently no evidence that the MPXI adds any additional benefit to differentiate sepsis from non-infectious SIRS in the ED. Therefore, further study will be needed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  13. Impact of myeloperoxidase-derived oxidants on the product profile of human 5-lipoxygenase.

    PubMed

    Zschaler, Josefin; Dorow, Juliane; Schöpe, Louisa; Ceglarek, Uta; Arnhold, Jürgen

    2015-08-01

    Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6 E,8 Z,11 Z,14 Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl(-) system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in

  14. Titre and affinity of propylthiouracil-induced anti-myeloperoxidase antibodies are closely associated with the development of clinical vasculitis.

    PubMed

    Ye, Hua; Gao, Ying; Guo, Xiao-Hui; Zhao, Ming-Hui

    2005-10-01

    Substantial evidences suggested that propylthiouracil (PTU) could induced anti-myeloperoxidase (MPO) antibodies in sera from patients with hyperthyroidism, however, only a subgroup of the PTU-induced anti-MPO antibody positive patients developed clinical evident vasculitis. The aim of this study is to compare the titres and affinities of PTU induced anti-MPO antibodies in sera from patients with hyperthyroidism with and without clinical vasculitis. Anti-MPO antibody positive sera from patients diagnosed hyperthyroidism with (n = 13) and without (n = 14) clinical evident vasculitis were collected. The titre was determined by MPO-ELISA and expressed as logarithm value (lgT). The affinity constant (aK) of anti-MPO IgG was measured by antigen inhibition assay. The titre and aK values were compared between patients with and without vasculitis. In patients with vasculitis, the mean lgT of anti-MPO antibodies was 3.62 +/- 0.66; the median aK was 4.47 x 10(7)M(-1). In patients without vasculitis, the mean lgT was 2.54 +/- 0.29; the median aK was 0.14 x 10(7)M(-1), and both were significant lower than those in patients with vasculitis (t = 5.464; P = 0.000 & z = -4.373; P = 0.000, respectively). We concluded that the titre and affinity of anti-MPO antibodies might be associated with the development of clinical vasculitis in patients with PTU-induced ANCA.

  15. Use of a spatial scan statistic to identify clusters of births occurring outside Ghanaian health facilities for targeted intervention.

    PubMed

    Bosomprah, Samuel; Dotse-Gborgbortsi, Winfred; Aboagye, Patrick; Matthews, Zoe

    2016-11-01

    To identify and evaluate clusters of births that occurred outside health facilities in Ghana for targeted intervention. A retrospective study was conducted using a convenience sample of live births registered in Ghanaian health facilities from January 1 to December 31, 2014. Data were extracted from the district health information system. A spatial scan statistic was used to investigate clusters of home births through a discrete Poisson probability model. Scanning with a circular spatial window was conducted only for clusters with high rates of such deliveries. The district was used as the geographic unit of analysis. The likelihood P value was estimated using Monte Carlo simulations. Ten statistically significant clusters with a high rate of home birth were identified. The relative risks ranged from 1.43 ("least likely" cluster; P=0.001) to 1.95 ("most likely" cluster; P=0.001). The relative risks of the top five "most likely" clusters ranged from 1.68 to 1.95; these clusters were located in Ashanti, Brong Ahafo, and the Western, Eastern, and Greater regions of Accra. Health facility records, geospatial techniques, and geographic information systems provided locally relevant information to assist policy makers in delivering targeted interventions to small geographic areas. Copyright © 2016 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

  16. Network analysis of translocated Takahe populations to identify disease surveillance targets.

    PubMed

    Grange, Zoë L; VAN Andel, Mary; French, Nigel P; Gartrell, Brett D

    2014-04-01

    network in 2011. Likewise, the wild Murchison Mountains population was consistently the sink of the network. Other nodes, such as the offshore islands and the wildlife hospital, varied in importance over time. Common network descriptors and measures of centrality identified key locations for targeting disease surveillance. The visual representation of movements of animals in a population that this technique provides can aid decision makers when they evaluate translocation proposals or attempt to control a disease outbreak. © 2014 Society for Conservation Biology.

  17. Identifying water price and population criteria for meeting future urban water demand targets

    NASA Astrophysics Data System (ADS)

    Ashoori, Negin; Dzombak, David A.; Small, Mitchell J.

    2017-12-01

    Predictive models for urban water demand can help identify the set of factors that must be satisfied in order to meet future targets for water demand. Some of the explanatory variables used in such models, such as service area population and changing temperature and rainfall rates, are outside the immediate control of water planners and managers. Others, such as water pricing and the intensity of voluntary water conservation efforts, are subject to decisions and programs implemented by the water utility. In order to understand this relationship, a multiple regression model fit to 44 years of monthly demand data (1970-2014) for Los Angeles, California was applied to predict possible future demand through 2050 under alternative scenarios for the explanatory variables: population, price, voluntary conservation efforts, and temperature and precipitation outcomes predicted by four global climate models with two CO2 emission scenarios. Future residential water demand in Los Angeles is projected to be largely driven by price and population rather than climate change and conservation. A median projection for the year 2050 indicates that residential water demand in Los Angeles will increase by approximately 36 percent, to a level of 620 million m3 per year. The Monte Carlo simulations of the fitted model for water demand were then used to find the set of conditions in the future for which water demand is predicted to be above or below the Los Angeles Department of Water and Power 2035 goal to reduce residential water demand by 25%. Results indicate that increases in price can not ensure that the 2035 water demand target can be met when population increases. Los Angeles must rely on furthering their conservation initiatives and increasing their use of stormwater capture, recycled water, and expanding their groundwater storage. The forecasting approach developed in this study can be utilized by other cities to understand the future of water demand in water-stressed areas

  18. Identifying clusters of falls-related hospital admissions to inform population targets for prioritising falls prevention programmes

    PubMed Central

    Finch, Caroline F; Stephan, Karen; Shee, Anna Wong; Hill, Keith; Haines, Terry P; Clemson, Lindy; Day, Lesley

    2015-01-01

    Background There has been limited research investigating the relationship between injurious falls and hospital resource use. The aims of this study were to identify clusters of community-dwelling older people in the general population who are at increased risk of being admitted to hospital following a fall and how those clusters differed in their use of hospital resources. Methods Analysis of routinely collected hospital admissions data relating to 45 374 fall-related admissions in Victorian community-dwelling older adults aged ≥65 years that occurred during 2008/2009 to 2010/2011. Fall-related admission episodes were identified based on being admitted from a private residence to hospital with a principal diagnosis of injury (International Classification of Diseases (ICD)-10-AM codes S00 to T75) and having a first external cause of a fall (ICD-10-AM codes W00 to W19). A cluster analysis was performed to identify homogeneous groups using demographic details of patients and information on the presence of comorbidities. Hospital length of stay (LOS) was compared across clusters using competing risks regression. Results Clusters based on area of residence, demographic factors (age, gender, marital status, country of birth) and the presence of comorbidities were identified. Clusters representing hospitalised fallers with comorbidities were associated with longer LOS compared with other cluster groups. Clusters delineated by demographic factors were also associated with increased LOS. Conclusions All patients with comorbidity, and older women without comorbidities, stay in hospital longer following a fall and hence consume a disproportionate share of hospital resources. These findings have important implications for the targeting of falls prevention interventions for community-dwelling older people. PMID:25618735

  19. Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion*

    PubMed Central

    Hung, Chien-Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

    2016-01-01

    Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. PMID:27477394

  20. IDENTIFYING AND TARGETING TUMOR-INITIATING CELLS IN THE TREATMENT OF BREAST CANCER

    PubMed Central

    Wei, Wei; Lewis, Michael T.

    2015-01-01

    Breast cancer is the most common cancer in women (exclusive of skin cancer), and is the second leading cause of cancer-related deaths. Although conventional and targeted therapies have improved survival rates, there are still considerable challenges in treating breast cancer, including treatment resistance, disease recurrence, and metastasis. Treatment resistance can be either de novo - due to traits that tumor cells possess prior to treatment, or acquired, - due to traits that tumor cells gain in response to treatment. A recently proposed mechanism of de novo resistance invokes existence of a specialized subset of cancer cells defined as tumor-initiating cells (TICs), or cancer stem cells (CSC). TICs have the capacity to self-renew and regenerate new tumors that consist of all clonally-derived cell types present in the parental tumor. There are data to suggest that TICs are resistant to many conventional cancer therapies, and survive treatment in spite of dramatic shrinkage of the tumor. Residual TICs can then eventually regrow resulting in disease relapse. It is also hypothesized that TIC may be responsible for metastatic disease. If these hypotheses are correct, targeting TICs may be imperative to achieve cure. In this review, we discuss evidence for breast TICs and their apparent resistance to conventional chemotherapy and radiotherapy, as well as to various targeted therapies. We also address the potential impact of breast TIC plasticity and metastatic potential on therapeutic strategies. Finally, we describe several genes and signaling pathways that appear important for TIC function that may represent promising therapeutic targets. PMID:25876646

  1. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    PubMed

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  2. Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer

    PubMed Central

    Searleman, Adam C.; Iliuk, Anton B.; Collier, Timothy S.; Chodosh, Lewis A.; Tao, W. Andy; Bose, Ron

    2014-01-01

    Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer-based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2-driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC-MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC-MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well-known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA-approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens. PMID:24723360

  3. Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells.

    PubMed

    Wenzel, Uwe; Herzog, Angelika; Kuntz, Sabine; Daniel, Hannelore

    2004-07-01

    A high dietary intake of plant foods is thought to contribute to the prevention of colorectal cancers in humans and flavonoids as part of such a diet are considered to contribute to those protective effects. Quercetin is a major dietary flavonoid consumed with a diet rich in onions, tea, and apples. We used HT-29 human colon cancer cells and investigated the effects of quercetin on proliferation, apoptosis, and differentiation as processes shown to be disregulated during cancer development. To identify the cellular targets of quercetin action, two-dimensional gel electrophoresis was performed and proteins altered in expression level after quercetin exposure of cells were identified by mass spectrometry of peptide fragments generated by tryptic digestion. Quercetin inhibited the proliferation of HT-29 cells with an IC(50)-value of 81.2 +/- 6.6 microM. Cell differentiation based on surface expression of alkaline phosphatase was enhanced 4-fold and the activity of the pro-apoptotic effector caspase-3 increased 3-fold. Those effects were associated with the regulation of heat-shock proteins and annexins shown to both play a crucial role in the process of apoptosis. Cytoskeletal caspase substrates were found as regulated as well and various proteins involved in intermediary metabolism and in gene regulation showed altered steady-state expression levels upon quercetin treatment of cells. In conclusion, quercetin alters the levels of a variety of proteins involved in growth, differentiation, and apoptosis of colon cancer cells. Their identification as molecular targets of quercetin may explain the anti-cancer activities of this flavonoid.

  4. Functional profiling of receptor tyrosine kinases and downstream signaling in human chondrosarcomas identifies pathways for rational targeted therapy.

    PubMed

    Zhang, Yi-Xiang; van Oosterwijk, Jolieke G; Sicinska, Ewa; Moss, Samuel; Remillard, Stephen P; van Wezel, Tom; Bühnemann, Claudia; Hassan, Andrew B; Demetri, George D; Bovée, Judith V M G; Wagner, Andrew J

    2013-07-15

    Chondrosarcomas are notoriously resistant to cytotoxic chemotherapeutic agents. We sought to identify critical signaling pathways that contribute to their survival and proliferation, and which may provide potential targets for rational therapeutic interventions. Activation of receptor tyrosine kinases (RTK) was surveyed using phospho-RTK arrays. S6 phosphorylation and NRAS mutational status were examined in chondrosarcoma primary tumor tissues. siRNA or small-molecule inhibitors against RTKs or downstream signaling proteins were applied to chondrosarcoma cells and changes in biochemical signaling, cell cycle, and cell viability were determined. In vivo antitumor activity of BEZ235, a phosphoinositide 3-kinase (PI3K)/mTOR inhibitor, was evaluated in a chondrosarcoma xenograft model. Several RTKs were identified as critical mediators of cell growth, but the RTK dependencies varied among cell lines. In exploration of downstream signaling pathways, strong S6 phosphorylation was found in 69% of conventional chondrosarcomas and 44% of dedifferentiated chondrosarcomas. Treatment with BEZ235 resulted in dramatic reduction in the growth of all chondrosarcoma cell lines. Tumor growth was similarly inhibited in a xenograft model of chondrosarcoma. In addition, chondrosarcoma cells with an NRAS mutation were sensitive to treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor. Functional NRAS mutations were found in 12% of conventional central chondrosarcomas. RTKs are commonly activated in chondrosarcoma, but because of their considerable heterogeneity, targeted inhibition of the PI3K/mTOR pathway represents a rational therapeutic strategy. Chondrosarcomas with NRAS mutations may benefit from treatment with MEK inhibitors.

  5. A Targeted RNAi Screen Identifies Endocytic Trafficking Factors That Control GLP-1 Receptor Signaling in Pancreatic β-Cells.

    PubMed

    Buenaventura, Teresa; Kanda, Nisha; Douzenis, Phoebe C; Jones, Ben; Bloom, Stephen R; Chabosseau, Pauline; Corrêa, Ivan R; Bosco, Domenico; Piemonti, Lorenzo; Marchetti, Piero; Johnson, Paul R; Shapiro, A M James; Rutter, Guy A; Tomas, Alejandra

    2018-03-01

    The glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) is a key target for type 2 diabetes (T2D) treatment. Because endocytic trafficking of agonist-bound receptors is one of the most important routes for regulation of receptor signaling, a better understanding of this process may facilitate the development of new T2D therapeutic strategies. Here, we screened 29 proteins with known functions in G protein-coupled receptor trafficking for their role in GLP-1R potentiation of insulin secretion in pancreatic β-cells. We identify five (clathrin, dynamin1, AP2, sorting nexins [SNX] SNX27, and SNX1) that increase and four (huntingtin-interacting protein 1 [HIP1], HIP14, GASP-1, and Nedd4) that decrease insulin secretion from murine insulinoma MIN6B1 cells in response to the GLP-1 analog exendin-4. The roles of HIP1 and the endosomal SNX1 and SNX27 were further characterized in mouse and human β-cell lines and human islets. While HIP1 was required for the coupling of cell surface GLP-1R activation with clathrin-dependent endocytosis, the SNXs were found to control the balance between GLP-1R plasma membrane recycling and lysosomal degradation and, in doing so, determine the overall β-cell incretin responses. We thus identify key modulators of GLP-1R trafficking and signaling that might provide novel targets to enhance insulin secretion in T2D. © 2017 by the American Diabetes Association.

  6. Increasing organizational energy conservation behaviors: Comparing the theory of planned behavior and reasons theory for identifying specific motivational factors to target for change

    NASA Astrophysics Data System (ADS)

    Finlinson, Scott Michael

    Social scientists frequently assess factors thought to underlie behavior for the purpose of designing behavioral change interventions. Researchers commonly identify these factors by examining relationships between specific variables and the focal behaviors being investigated. Variables with the strongest relationships to the focal behavior are then assumed to be the most influential determinants of that behavior, and therefore often become the targets for change in a behavioral change intervention. In the current proposal, multiple methods are used to compare the effectiveness of two theoretical frameworks for identifying influential motivational factors. Assessing the relative influence of all factors and sets of factors for driving behavior should clarify which framework and methodology is the most promising for identifying effective change targets. Results indicated each methodology adequately predicted the three focal behaviors examined. However, the reasons theory approach was superior for predicting factor influence ratings compared to the TpB approach. While common method variance contamination had minimal impact on the results or conclusions derived from the present study's findings, there were substantial differences in conclusions depending on the questionnaire design used to collect the data. Examples of applied uses of the present study are discussed.

  7. Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis.

    PubMed

    Kulkeaw, Kasem; Inoue, Tomoko; Ishitani, Tohru; Nakanishi, Yoichi; Zon, Leonard I; Sugiyama, Daisuke

    2018-02-01

    Zebrafish embryos are useful to study haematopoietic gene function in vertebrates, although lack of antibodies to zebrafish proteins has limited the purification of specific cell populations. Here, we purified primitive zebrafish erythrocytes using 1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione (DRAQ5 TM ), a DNA-staining fluorescent dye. At 48-h post-fertilization, we sorted small-sized cells from embryos using forward scatter and found that they consisted of DRAQ5 high and DRAQ5 low populations. DRAQ5 high cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin (hbae3 and hbbe1.1) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5 TM analysis of gata1:dsRed transgenic embryos, we purified primitive DRAQ5 high dsRed+ erythrocytes from haematopoietic progenitor cells. Using this method, we identified docking protein 2 (Dok2) as functioning in differentiation of primitive erythrocytes. We conclude that DRAQ5 TM -based flow cytometry enables purification of primitive zebrafish erythrocytes. © 2017 John Wiley & Sons Ltd.

  8. The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn's Disease-Like Ileitis.

    PubMed

    Rodriguez-Palacios, Alexander; Harding, Andrew; Menghini, Paola; Himmelman, Catherine; Retuerto, Mauricio; Nickerson, Kourtney P; Lam, Minh; Croniger, Colleen M; McLean, Mairi H; Durum, Scott K; Pizarro, Theresa T; Ghannoum, Mahmoud A; Ilic, Sanja; McDonald, Christine; Cominelli, Fabio

    2018-04-23

    Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn's disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice. Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation. Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene-carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice. Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal

  9. Effects of paraoxonase, arylesterase, ceruloplasmin, catalase, and myeloperoxidase activities on prognosis in pediatric patients with sepsis.

    PubMed

    Ayar, Ganime; Atmaca, Yasemin Men; Alışık, Murat; Erel, Özcan

    2017-05-01

    The present study aimed to investigate the levels of paraoxonase (PON), stimulated paraoxonase (SPON), arylesterase (ARE), ceruloplasmin (CLP), myeloperoxidase (MPO), and catalase (CAT) in pediatric sepsis and to explore their effects on the prognosis of sepsis. Patients diagnosed with sepsis (n=33) and healthy controls (n=30) were included. PON, SPON, ARE, CLP, MPO, and CAT activities were measured in the sepsis and control groups. Additionally, the parameters were compared between survivors and non-survivors in the sepsis group. The levels of hemoglobin, white blood cell, platelet, lactate, and C-reactive protein were measured in the blood samples drawn from the patients with sepsis at diagnosis, at the 48th hour, and on day 7. The pediatric risk of mortality and pediatric logistic organ dysfunction scores of the patients were used for the estimation of severity of disease. Lower ARE (153.24 vs. 264.32U/L; p<0.001), lower CLP (80.58 vs. 97.98U/L; p=0.032), lower MPO (91.24 vs. 116.55U/L; p=0.023), and higher CAT levels (256.5 vs.145.5kU/L; p=0.003) were determined in the sepsis group as compared to the control group. There was no difference between the groups in terms of PON or SPON levels. No difference was determined between the survivors and non-survivors in terms of any of the parameters. The present study determined that ARE, CLP, CAT, and MPO levels are different between the pediatric patients with sepsis and healthy controls. ARE level can be a potent biomarker for sepsis in critical patients in intensive care units. Further studies with larger samples are required to demonstrate the value of these parameters as prognostic biomarkers in pediatric sepsis. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  10. Modes of targets in water excited and identified using radiation pressure of modulated focused ultrasound

    NASA Astrophysics Data System (ADS)

    Daniel, Timothy; Fortuner, Auberry; Abawi, Ahmad; Kirsteins, Ivars; Marston, Philip

    2016-11-01

    The modulated radiation pressure (MRP) of ultrasound has been widely used to selectively excite low frequency modes of fluid objects. We previously used MRP to excite less compliant metallic object in water including the low frequency modes of a circular metal plate in water. A larger focused ultrasonic transducer allows us to drive modes of larger more-realistic targets. In our experiments solid targets are suspended by strings or supported on sand and the modulated ultrasound is focused on the target's surface. Target sound emissions were recorded and a laser vibrometer was used to measure the surface velocity of the target to give the magnitude of the target response. The source transducer was driven with a doublesideband suppressed carrier voltage as in. By varying the modulation frequency and monitoring target response, resonant frequencies can be measured and compared to finite element models. We also demonstrate the radiation torque of a focused first-order acoustic vortex beam associated with power absorption in the Stokes layer adjacent to a sphere. Funded by ONR.

  11. Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD.

    PubMed

    Sabbagh, Jonathan J; Cordova, Ricardo A; Zheng, Dali; Criado-Marrero, Marangelie; Lemus, Andrea; Li, Pengfei; Baker, Jeremy D; Nordhues, Bryce A; Darling, April L; Martinez-Licha, Carlos; Rutz, Daniel A; Patel, Shreya; Buchner, Johannes; Leahy, James W; Koren, John; Dickey, Chad A; Blair, Laura J

    2018-06-19

    Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.

  12. A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

    PubMed

    Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

    2014-08-08

    Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing

  13. Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

    PubMed

    Viringipurampeer, Ishaq A; Gregory-Evans, Cheryl Y; Metcalfe, Andrew L; Bashar, Emran; Moritz, Orson L; Gregory-Evans, Kevin

    2018-06-18

    Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

  14. Functional Genomics to Identify Therapeutic Targets in Cancer Stem Cells Using a Novel Murine CRPC Model

    DTIC Science & Technology

    2015-11-01

    REPORT 3 . DATES COVERED 6 Aug 2013 - 5 Aug 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Functional Genomics to Identify Therapeutic Targets in...4 3 . Accomplishments………..………………………………...…………...4 4. Impact…………………………...……………………….….…………21 5. Changes/Problems...Requirements……………………….…….………23 9. Appendices……………………………………………….…….………23 3 1. INTRODUCTION: Prostate cancer is the most common noncutaneous malignancy in men

  15. Nuclear Security: Target Analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Singh, Surinder Paul; Gibbs, Philip W.; Bultz, Garl A.

    2014-03-01

    This objectives of this session were to understand the basic steps of target identification; describe the SNRI targets in detail; characterize specific targets with more detail; prioritize targets based on guidance documents; understand the graded safeguards concept; identify roll up and understand why it is a concern; and recognize the category for different materials.

  16. Myeloperoxidase-derived chlorinating species induce protein carbamylation through decomposition of thiocyanate and urea: Novel pathways generating dysfunctional high-density lipoprotein

    PubMed Central

    Holzer, Michael; Zangger, Klaus; El-Gamal, Dalia; Binder, Veronika; Curcic, Sanja; Konya, Viktoria; Schuligoi, Rufina; Heinemann, Akos; Marsche, Gunther

    2013-01-01

    Aim Protein carbamylation through cyanate is thought to have a causal role in promoting cardiovascular disease. We recently observed that the phagocyte protein myeloperoxidase (MPO) specifically induces high-density lipoprotein carbamylation, rather than chlorination, in human atherosclerotic lesions, raising the possibility that MPO-derived chlorinating species are involved in cyanate formation. Results Here we show that MPO-derived chlorinating species rapidly decompose the plasma components thiocyanate and urea thereby promoting (lipo)protein carbamylation. Strikingly, the presence of physiologic concentrations of thiocyanate completely prevented MPO-induced 3-chlorotyrosine formation in HDL. Moreover, thiocyanate scavenged a 2.5-fold molar excess of hypochlorous acid, promoting HDL carbamylation, but not chlorination. Carbamylation of HDL resulted in a loss of anti-inflammatory and anti-oxidative properties. Cyanate significantly impaired (i) HDL’s ability to activate lecithin-cholesterol acyltransferase, (ii) the activity of paraoxonase, a major HDL-associated anti-inflammatory enzyme and (iii) the anti-oxidative activity of HDL. Innovation Here we report that MPO-derived chlorinating species preferentially induce protein carbamylation - rather than chlorination - in the presence of physiologically relevant thiocyanate concentrations. Carbamylation of HDL results in the loss of its anti-inflammatory and anti-oxidative activities. Conclusion MPO-mediated decomposition of thiocyanate and/or urea might be a relevant mechanism for generating dysfunctional HDL in human disease. PMID:22462773

  17. A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

    PubMed

    Vij, Rajesh; Lin, Zhonghua; Chiang, Nancy; Vernes, Jean-Michel; Storek, Kelly M; Park, Summer; Chan, Joyce; Meng, Y Gloria; Comps-Agrar, Laetitia; Luan, Peng; Lee, Sophia; Schneider, Kellen; Bevers, Jack; Zilberleyb, Inna; Tam, Christine; Koth, Christopher M; Xu, Min; Gill, Avinash; Auerbach, Marcy R; Smith, Peter A; Rutherford, Steven T; Nakamura, Gerald; Seshasayee, Dhaya; Payandeh, Jian; Koerber, James T

    2018-05-08

    Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

  18. A method for rapid, targeted CNV genotyping identifies rare variants associated with neurocognitive disease.

    PubMed

    Mefford, Heather C; Cooper, Gregory M; Zerr, Troy; Smith, Joshua D; Baker, Carl; Shafer, Neil; Thorland, Erik C; Skinner, Cindy; Schwartz, Charles E; Nickerson, Deborah A; Eichler, Evan E

    2009-09-01

    Copy-number variants (CNVs) are substantial contributors to human disease. A central challenge in CNV-disease association studies is to characterize the pathogenicity of rare and possibly incompletely penetrant events, which requires the accurate detection of rare CNVs in large numbers of individuals. Cost and throughput issues limit our ability to perform these studies. We have adapted the Illumina BeadXpress SNP genotyping assay and developed an algorithm, SNP-Conditional OUTlier detection (SCOUT), to rapidly and accurately detect both rare and common CNVs in large cohorts. This approach is customizable, cost effective, highly parallelized, and largely automated. We applied this method to screen 69 loci in 1105 children with unexplained intellectual disability, identifying pathogenic variants in 3.1% of these individuals and potentially pathogenic variants in an additional 2.3%. We identified seven individuals (0.7%) with a deletion of 16p11.2, which has been previously associated with autism. Our results widen the phenotypic spectrum of these deletions to include intellectual disability without autism. We also detected 1.65-3.4 Mbp duplications at 16p13.11 in 1.1% of affected individuals and 350 kbp deletions at 15q11.2, near the Prader-Willi/Angelman syndrome critical region, in 0.8% of affected individuals. Compared to published CNVs in controls they are significantly (P = 4.7 x 10(-5) and 0.003, respectively) enriched in these children, supporting previously published hypotheses that they are neurocognitive disease risk factors. More generally, this approach offers a previously unavailable balance between customization, cost, and throughput for analysis of CNVs and should prove valuable for targeted CNV detection in both research and diagnostic settings.

  19. Drug-Target Interactions: Prediction Methods and Applications.

    PubMed

    Anusuya, Shanmugam; Kesherwani, Manish; Priya, K Vishnu; Vimala, Antonydhason; Shanmugam, Gnanendra; Velmurugan, Devadasan; Gromiha, M Michael

    2018-01-01

    Identifying the interactions between drugs and target proteins is a key step in drug discovery. This not only aids to understand the disease mechanism, but also helps to identify unexpected therapeutic activity or adverse side effects of drugs. Hence, drug-target interaction prediction becomes an essential tool in the field of drug repurposing. The availability of heterogeneous biological data on known drug-target interactions enabled many researchers to develop various computational methods to decipher unknown drug-target interactions. This review provides an overview on these computational methods for predicting drug-target interactions along with available webservers and databases for drug-target interactions. Further, the applicability of drug-target interactions in various diseases for identifying lead compounds has been outlined. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    PubMed Central

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

    2015-01-01

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

  1. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

    2016-02-12

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative tomore » other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.« less

  2. Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

    PubMed

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

    2016-01-01

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  3. Identifying therapeutic targets in gastric cancer: the current status and future direction

    PubMed Central

    Yu, Beiqin; Xie, Jingwu

    2016-01-01

    Gastric cancer is the third leading cause of cancer-related death worldwide. Our basic understanding of gastric cancer biology falls behind that of many other cancer types. Current standard treatment options for gastric cancer have not changed for the last 20 years. Thus, there is an urgent need to establish novel strategies to treat this deadly cancer. Successful clinical trials with Gleevec in CML and gastrointestinal stromal tumors have set up an example for targeted therapy of cancer. In this review, we will summarize major progress in classification, therapeutic options of gastric cancer. We will also discuss molecular mechanisms for drug resistance in gastric cancer. In addition, we will attempt to propose potential future directions in gastric cancer biology and drug targets. PMID:26373844

  4. Novel mutations in CRB1 gene identified in a chinese pedigree with retinitis pigmentosa by targeted capture and next generation sequencing

    PubMed Central

    Lo, David; Weng, Jingning; Liu, xiaohong; Yang, Juhua; He, Fen; Wang, Yun; Liu, Xuyang

    2016-01-01

    PURPOSE To detect the disease-causing gene in a Chinese pedigree with autosomal-recessive retinitis pigmentosa (ARRP). METHODS All subjects in this family underwent a complete ophthalmic examination. Targeted-capture next generation sequencing (NGS) was performed on the proband to detect variants. All variants were verified in the remaining family members by PCR amplification and Sanger sequencing. RESULTS All the affected subjects in this pedigree were diagnosed with retinitis pigmentosa (RP). The compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations in the Crumbs homolog 1 (CRB1) gene were identified in all the affected patients but not in the unaffected individuals in this family. These mutations were inherited from their parents, respectively. CONCLUSION The novel compound heterozygous mutations in CRB1 were identified in a Chinese pedigree with ARRP using targeted-capture next generation sequencing. After evaluating the significant heredity and impaired protein function, the compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations are the causal genes of early onset ARRP in this pedigree. To the best of our knowledge, there is no previous report regarding the compound mutations. PMID:27806333

  5. Large Dog Relinquishment to Two Municipal Facilities in New York City and Washington, D.C.: Identifying Targets for Intervention

    PubMed Central

    Weiss, Emily; Slater, Margaret; Garrison, Laurie; Drain, Natasha; Dolan, Emily; Scarlett, Janet M.; Zawistowski, Stephen L.

    2014-01-01

    Simple Summary While the overall trend in euthanasia has been decreasing nationally, large dogs are at a higher risk of euthanasia than other-sized dogs in most animal shelters in the United States. We hypothesized that one way to increase the lives saved with regard to large dogs in shelters is to keep them home in the first place when possible. Our research is the first to collect data in New York City and Washington, D.C., identifying the process leading to the owner relinquishment of large dogs. We found that targets for interventions to decrease large dog relinquishment are likely different in each community. Abstract While the overall trend in euthanasia has been decreasing nationally, large dogs are at a higher risk of euthanasia than other sized dogs in most animal shelters in the United States. We hypothesized one way to increase the lives saved with respect to these large dogs is to keep them home when possible. In order to develop solutions to decrease relinquishment, a survey was developed to learn more about the reasons owners relinquish large dogs. The survey was administered to owners relinquishing their dogs at two large municipal facilities, one in New York City and one in Washington, D.C. There were 157 responses between the two facilities. We found both significant similarities and differences between respondents and their dogs from the two cities. We identified opportunities to potentially support future relinquishers and found that targets for interventions are likely different in each community. PMID:26480315

  6. Candidate adaptive genes associated with lineage divergence: identifying SNPs via next-generation targeted resequencing in mule deer (Odocoileus hemionus).

    PubMed

    Powell, John H; Amish, Stephen J; Haynes, Gwilym D; Luikart, Gordon; Latch, Emily K

    2016-09-01

    Mule deer (Odocoileus hemionus) are an excellent nonmodel species for empirically testing hypotheses in landscape and population genomics due to their large population sizes (low genetic drift), relatively continuous distribution, diversity of occupied habitats and phenotypic variation. Because few genomic resources are currently available for this species, we used exon data from a cattle (Bos taurus) reference genome to direct targeted resequencing of 5935 genes in mule deer. We sequenced approximately 3.75 Mbp at minimum 20X coverage in each of the seven mule deer, identifying 23 204 single nucleotide polymorphisms (SNPs) within, or adjacent to, 6886 exons in 3559 genes. We found 91 SNP loci (from 69 genes) with putatively fixed allele frequency differences between the two major lineages of mule deer (mule deer and black-tailed deer), and our estimate of mean genetic divergence (genome-wide FST  = 0.123) between these lineages was consistent with previous findings using microsatellite loci. We detected an over-representation of gamete generation and amino acid transport genes among the genes with SNPs exhibiting potentially fixed allele frequency differences between lineages. This targeted resequencing approach using exon capture techniques has identified a suite of loci that can be used in future research to investigate the genomic basis of adaptation and differentiation between black-tailed deer and mule deer. This study also highlights techniques (and an exon capture array) that will facilitate population genomic research in other cervids and nonmodel organisms. © 2016 John Wiley & Sons Ltd.

  7. Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

    PubMed Central

    2010-01-01

    Background Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production. Results In the absence of anatomical constraints to digestion, lignification with normal monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree of ester group hydroxylation

  8. A systems approach to identifying correlated gene targets for the loss of colour pigmentation in plants

    PubMed Central

    2011-01-01

    Background The numerous diverse metabolic pathways by which plant compounds can be produced make it difficult to predict how colour pigmentation is lost for different tissues and plants. This study employs mathematical and in silico methods to identify correlated gene targets for the loss of colour pigmentation in plants from a whole cell perspective based on the full metabolic network of Arabidopsis. This involves extracting a self-contained flavonoid subnetwork from the AraCyc database and calculating feasible metabolic routes or elementary modes (EMs) for it. Those EMs leading to anthocyanin compounds are taken to constitute the anthocyanin biosynthetic pathway (ABP) and their interplay with the rest of the EMs is used to study the minimal cut sets (MCSs), which are different combinations of reactions to block for eliminating colour pigmentation. By relating the reactions to their corresponding genes, the MCSs are used to explore the phenotypic roles of the ABP genes, their relevance to the ABP and the impact their eliminations would have on other processes in the cell. Results Simulation and prediction results of the effect of different MCSs for eliminating colour pigmentation correspond with existing experimental observations. Two examples are: i) two MCSs which require the simultaneous suppression of genes DFR and ANS to eliminate colour pigmentation, correspond to observational results of the same genes being co-regulated for eliminating floral pigmentation in Aquilegia and; ii) the impact of another MCS requiring CHS suppression, corresponds to findings where the suppression of the early gene CHS eliminated nearly all flavonoids but did not affect the production of volatile benzenoids responsible for floral scent. Conclusions From the various MCSs identified for eliminating colour pigmentation, several correlate to existing experimental observations, indicating that different MCSs are suitable for different plants, different cells, and different conditions

  9. Altruistic behavior in cohesive social groups: The role of target identifiability.

    PubMed

    Ritov, Ilana; Kogut, Tehila

    2017-01-01

    People's tendency to be more generous toward identifiable victims than toward unidentifiable or statistical victims is known as the Identifiable Victim Effect. Recent research has called the generality of this effect into question, showing that in cross-national contexts, identifiability mostly affects willingness to help victims of one's own "in-group." Furthermore, in inter-group conflict situations, identifiability increased generosity toward a member of the adversary group, but decreased generosity toward a member of one's own group. In the present research we examine the role of group-cohesiveness as an underlying factor accounting for these divergent findings. In particular, we examined novel groups generated in the lab, using the minimal group paradigm, as well as natural groups of students in regular exercise sections. Allocation decisions in dictator games revealed that a group's cohesiveness affects generosity toward in-group and out-group recipients differently, depending on their identifiability. In particular, in cohesive groups the identification of an in-group recipient decreased, rather than increased generosity.

  10. Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

    PubMed Central

    Maitra, Dhiman; Shaeib, Faten; Abdulhamid, Ibrahim; Abdulridha, Rasha M.; Saed, Ghassan M.; Diamond, Michael P.; Pennathur, Subramaniam; Abu-Soud, Husam M.

    2013-01-01

    Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl−) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV–visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10 μM), precluding the enzyme from functioning at maximum activity (80–90% inhibition). MPO heme destruction occurred only in the presence of Cl−. Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO–Compound I–Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron. PMID:23624305

  11. Use of surveillance data to identify target populations for Staphylococcus aureus vaccines and prevent surgical site infections: A pilot study

    PubMed Central

    Gustin, Marie-Paule; Giard, Marine; Bénet, Thomas; Vanhems, Philippe

    2015-01-01

    The development of anti-staphylococcal vaccines is nowadays a priority to prevent surgical site infections (SSI). The objective of the present study was to identify a potential target population by assessing surveillance data on surgery patients for possible anti-staphylococcal vaccine administration. Individuals at high risk of SSI by Staphylococcus aureus (SA) were targeted by the French SSI Surveillance Network in south-eastern France between 2008 and 2011. Among 238,470 patients, those undergoing primary total hip replacement appeared to be an interesting and healthy enough population for anti-staphylococcal vaccine testing. These male patients, subjected to multiple procedures and with American Society of Anesthesiologists score >2, had a probability of SA SSI about 21 times higher than females with no severe systemic disease and no multiple procedures. Our study indicates that surveillance data on SSI might be an interesting epidemiological source for planning vaccine trials to prevent nosocomial infections. PMID:25668663

  12. Kinome expression profiling of human neuroblastoma tumors identifies potential drug targets for ultra high-risk patients.

    PubMed

    Russo, Roberta; Cimmino, Flora; Pezone, Lucia; Manna, Francesco; Avitabile, Marianna; Langella, Concetta; Koster, Jan; Casale, Fiorina; Raia, Maddalena; Viola, Giampietro; Fischer, Matthias; Iolascon, Achille; Capasso, Mario

    2017-10-01

    Neuroblastoma (NBL) accounts for >7% of malignancies in patients younger than 15 years. Low- and intermediate-risk patients exhibit excellent or good prognosis after treatment, whereas for high-risk (HR) patients, the estimated 5-year survival rates is still <40%. The ability to stratify HR patients that will not respond to standard treatment strategies is critical for informed treatment decisions. In this study, we have generated a specific kinome gene signature, named Kinome-27, which is able to identify a subset of HR-NBL tumors, named ultra-HR NBL, with highly aggressive clinical behavior that not adequately respond to standard treatments. We have demonstrated that NBL cell lines expressing the same kinome signature of ultra-HR tumors (ultra-HR-like cell lines) may be selectively targeted by the use of two drugs [suberoylanilide hydroxamic acid (SAHA) and Radicicol], and that the synergic combination of these drugs is able to block the ultra-HR-like cells in G2/M phase of cell cycle. The use of our signature in clinical practice will allow identifying patients with negative outcome, which would benefit from new and more personalized treatments. Preclinical in vivo studies are needed to consolidate the SAHA and Radicicol treatment in ultra-HR NBL patients. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer.

    PubMed

    Giamas, Georgios; Filipović, Aleksandra; Jacob, Jimmy; Messier, Walter; Zhang, Hua; Yang, Dongyun; Zhang, Wu; Shifa, Belul Assefa; Photiou, Andrew; Tralau-Stewart, Cathy; Castellano, Leandro; Green, Andrew R; Coombes, R Charles; Ellis, Ian O; Ali, Simak; Lenz, Heinz-Josef; Stebbing, Justin

    2011-06-01

    Therapies targeting estrogen receptor α (ERα, encoded by ESR1) have transformed the treatment of breast cancer. However, large numbers of women relapse, highlighting the need for the discovery of new regulatory targets modulating ERα pathways. An siRNA screen identified kinases whose silencing alters the estrogen response including those previously implicated in regulating ERα activity (such as mitogen-activated protein kinase and AKT). Among the most potent regulators was lemur tyrosine kinase-3 (LMTK3), for which a role has not previously been assigned. In contrast to other modulators of ERα activity, LMTK3 seems to have been subject to Darwinian positive selection, a noteworthy result given the unique susceptibility of humans to ERα+ breast cancer. LMTK3 acts by decreasing the activity of protein kinase C (PKC) and the phosphorylation of AKT (Ser473), thereby increasing binding of forkhead box O3 (FOXO3) to the ESR1 promoter. LMTK3 phosphorylated ERα, protecting it from proteasomal degradation in vitro. Silencing of LMTK3 reduced tumor volume in an orthotopic mouse model and abrogated proliferation of ERα+ but not ERα- cells, indicative of its role in ERα activity. In human cancers, LMTK3 abundance and intronic polymorphisms were significantly associated with disease-free and overall survival and predicted response to endocrine therapies. These findings yield insights into the natural history of breast cancer in humans and reveal LMTK3 as a new therapeutic target.

  14. Altruistic behavior in cohesive social groups: The role of target identifiability

    PubMed Central

    Ritov, Ilana; Kogut, Tehila

    2017-01-01

    People’s tendency to be more generous toward identifiable victims than toward unidentifiable or statistical victims is known as the Identifiable Victim Effect. Recent research has called the generality of this effect into question, showing that in cross-national contexts, identifiability mostly affects willingness to help victims of one’s own “in-group.” Furthermore, in inter-group conflict situations, identifiability increased generosity toward a member of the adversary group, but decreased generosity toward a member of one’s own group. In the present research we examine the role of group-cohesiveness as an underlying factor accounting for these divergent findings. In particular, we examined novel groups generated in the lab, using the minimal group paradigm, as well as natural groups of students in regular exercise sections. Allocation decisions in dictator games revealed that a group’s cohesiveness affects generosity toward in-group and out-group recipients differently, depending on their identifiability. In particular, in cohesive groups the identification of an in-group recipient decreased, rather than increased generosity. PMID:29161282

  15. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

    DOE PAGES

    Reilly, Morgann C.; Kim, Joonhoon; Lynn, Jed; ...

    2018-01-06

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers ofmore » heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. As a result, this strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.« less

  16. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

    DOE PAGES

    Reilly, Morgann C.; Kim, Joonhoon; Lynn, Jed; ...

    2018-01-06

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers ofmore » heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.« less

  17. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reilly, Morgann C.; Kim, Joonhoon; Lynn, Jed

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers ofmore » heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.« less

  18. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reilly, Morgann C.; Kim, Joonhoon; Lynn, Jed

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers ofmore » heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. As a result, this strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.« less

  19. Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

    PubMed

    Ulrich, Julia; Dao, Van Anh; Majumdar, Upalparna; Schmitt-Engel, Christian; Schwirz, Jonas; Schultheis, Dorothea; Ströhlein, Nadi; Troelenberg, Nicole; Grossmann, Daniela; Richter, Tobias; Dönitz, Jürgen; Gerischer, Lizzy; Leboulle, Gérard; Vilcinskas, Andreas; Stanke, Mario; Bucher, Gregor

    2015-09-03

    Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening. We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites. Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

  20. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

    PubMed

    Yoo, Dae-goon; Floyd, Madison; Winn, Matthew; Moskowitz, Samuel M; Rada, Balázs

    2014-08-01

    Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ.

    PubMed

    Xu, Lijuan; Liu, Zijian; Li, Yang; Yin, Chao; Hu, Yachen; Xie, Xiaolei; Li, Qiuchun; Jiao, Xinan

    2018-06-01

    Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90 fg/μl or 10 2 CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.

  2. Comparison of enzyme-linked immunosorbent assay and rapid chemiluminescent analyser in the detection of myeloperoxidase and proteinase 3 autoantibodies.

    PubMed

    Pucar, Phillippa A; Hawkins, Carolyn A; Randall, Katrina L; Li, Candice; McNaughton, Euan; Cook, Matthew C

    2017-06-01

    Antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3) are vital in the diagnosis and management of ANCA-associated vasculitis. A chemiluminescent immunoassay (CLIA; Quanta Flash) provides MPO and PR3 antibody results in 30 minutes, which is much faster than enzyme-linked immunosorbent assay (ELISA). We compared the performance of ELISA (Orgentec) and CLIA (Quanta Flash) for MPO and PR3 antibody quantitation on 303 samples, comprising 196 consecutive samples received in a single diagnostic laboratory over a 3 month period, and 107 samples collected from 42 known vasculitis patients over a 40 month period. We observed a correlation between both methods using spearman correlation coefficients (MPO, r s  = 0.63, p < 0.01; PR3, r s  = 0.69, p < 0.01). There was agreement between both methods in determining a positive or negative result. In the vasculitis cohort, CLIA performed well at clinically important stages of disease; diagnosis (eight samples all positive by both assays) and disease relapse (correlation for both MPO and PR3 antibody quantitation r s  = 0.84, p = 0.03 and r s  = 0.78, p < 0.01, respectively). Three samples were discordant at clinical relapse, testing positive by CLIA, including one high positive associated with relapse requiring a change in treatment. In summary, CLIA appears to be at least as accurate as ELISA for measurement of MPO and PR3 antibodies. Copyright © 2017. Published by Elsevier B.V.

  3. Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination

    PubMed Central

    Brooks, Jill M.; Long, Heather M.; Tierney, Rose J.; Shannon-Lowe, Claire; Leese, Alison M.; Fitzpatrick, Martin; Taylor, Graham S.; Rickinson, Alan B.

    2016-01-01

    Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three “first wave” proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that “first wave” antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design. PMID:27096949

  4. Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy-filtering and energy-dispersive spectroscopy-scanning transmission electron microscopy.

    PubMed

    Moriguchi, Keiichi

    2018-03-01

    When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O 2 - ). Nox-generated O 2 - is rapidly converted to other ROS. Free radical-forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy-filtering and energy-dispersive spectroscopy-scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H 2 O 2 generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3'-diaminobenzidine-tetrahydrochloride (DAB) and 0.01% H 2 O 2 . A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N). © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  5. Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

    DTIC Science & Technology

    2014-06-01

    Specifically, we combined the CRISPR genome editing system with a novel approach allowing efficient single cell cloning of Drosophila cells with the aim of...and culture these to produce cultures completely lacking wildtype sequence at the target locus. No robust methods existed to clone single Drosophila ...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . 65 samples that displayed synthetic lethality (15 genes) or synthetic

  6. Fab’-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis

    PubMed Central

    Hamed, Laroui; Emilie, Viennois; Xiao, Bo; Canup, Brandon S.; Duke, Geem; Denning, Timothy L.; Didier, Merlin

    2014-01-01

    Patients suffering from Inflammatory Bowel Disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab’ portion of the F4/80 Ab (Fab’-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab’-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab’-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab’-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab’-bearing NPs loaded with TNFα siRNA than without the Fab’-bearing. Grafting the Fab’-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab’-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages. PMID:24810114

  7. A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

    PubMed

    Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

    2018-06-11

    We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

  8. Setting Achievement Targets for School Children.

    ERIC Educational Resources Information Center

    Thanassoulis, Emmanuel

    1999-01-01

    Develops an approach for setting performance targets for schoolchildren, using data-envelopment analysis to identify benchmark pupils who achieve the best observed performance (allowing for contextual factors). These pupils' achievement forms the basis of targets estimated. The procedure also identifies appropriate role models for weaker students'…

  9. Transcriptome profiling of equine vitamin E deficient neuroaxonal dystrophy identifies upregulation of liver X receptor target genes

    PubMed Central

    Finno, Carrie J.; Bordbari, Matthew H.; Valberg, Stephanie J.; Lee, David; Herron, Josi; Hines, Kelly; Monsour, Tamer; Scott, Erica; Bannasch, Danika L.; Mickelson, James; Xu, Libin

    2016-01-01

    Specific spontaneous heritable neurodegenerative diseases have been associated with lower serum and cerebrospinal fluid α-tocopherol (α-TOH) concentrations. Equine neuroaxonal dystrophy (eNAD) has similar histologic lesions to human ataxia with vitamin E deficiency caused by mutations in the α-TOH transfer protein gene (TTPA). Mutations in TTPA are not present with eNAD and the molecular basis remains unknown. Given the neuropathologic phenotypic similarity of the conditions, we assessed the molecular basis of eNAD by global transcriptome sequencing of the cervical spinal cord. Differential gene expression analysis identified 157 significantly (FDR<0.05) dysregulated transcripts within the spinal cord of eNAD-affected horses. Statistical enrichment analysis identified significant downregulation of the ionotropic and metabotropic group III glutamate receptor, synaptic vesicle trafficking and cholesterol biosynthesis pathways. Gene co-expression analysis identified one module of upregulated genes significantly associated with the eNAD phenotype that included the liver X receptor (LXR) targets CYP7A1, APOE, PLTP and ABCA1. Validation of CYP7A1 and APOE dysregulation was performed in an independent biologic group and CYP7A1 was found to be additionally upregulated in the medulla oblongata of eNAD horses. Evidence of LXR activation supports a role for modulation of oxysterol-dependent LXR transcription factor activity by tocopherols. We hypothesize that the protective role of α-TOH in eNAD may reside in its ability to prevent oxysterol accumulation and subsequent activation of the LXR in order to decrease lipid peroxidation associated neurodegeneration. PMID:27751910

  10. Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway

    PubMed Central

    Zhang, Yan; Fan, Kai-Ji; Sun, Qiang; Chen, Ai-Zhong; Shen, Wen-Long; Zhao, Zhi-Hu; Zheng, Xiao-Fei; Yang, Xiao

    2012-01-01

    The transforming growth factor-β (TGF-β) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-β signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-β transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3′-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-β to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-β signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4. PMID:22821565

  11. Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development.

    PubMed

    Litholdo, Celso G; Parker, Benjamin L; Eamens, Andrew L; Larsen, Martin R; Cordwell, Stuart J; Waterhouse, Peter M

    2016-06-01

    Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development*

    PubMed Central

    Litholdo, Celso G.; Parker, Benjamin L.; Eamens, Andrew L.; Larsen, Martin R.; Cordwell, Stuart J.; Waterhouse, Peter M.

    2016-01-01

    Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. PMID:27067051

  13. Effects of the Mitochondria-Targeted Antioxidant Mitoquinone in Murine Acute Pancreatitis

    PubMed Central

    Wen, Li; Szatmary, Peter; Mukherjee, Rajarshi; Armstrong, Jane; Chvanov, Michael; Tepikin, Alexei V.; Murphy, Michael P.; Sutton, Robert; Criddle, David N.

    2015-01-01

    Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease. PMID:25878403

  14. Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis.

    PubMed

    Huang, Wei; Cash, Nicole; Wen, Li; Szatmary, Peter; Mukherjee, Rajarshi; Armstrong, Jane; Chvanov, Michael; Tepikin, Alexei V; Murphy, Michael P; Sutton, Robert; Criddle, David N

    2015-01-01

    Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.

  15. Alleviation of 5-fluorouracil-induced intestinal mucositis in rats by vitamin E via targeting oxidative stress and inflammatory markers.

    PubMed

    Al-Asmari, Abdulrahman Khazim; Khan, Abdul Quaiyoom; Al-Asmari, Sarah A; Al-Rawi, Abdulqadir; Al-Omani, Saud

    2016-12-01

    BackgroundIntestinal mucositis is a major concern related with cancer therapy. It is well established that overproduction of reactive oxygen species and inflammatory mediators plays vital role in the pathogenesis of mucositis. The aim of the study was to investigate the modulatory effect of vitamin E (vit. E) on 5-fluorouracil (5-FU)-induced intestinal mucositis by targeting oxidative stress and inflammatory markers in rats. MethodsRats were randomly divided into four groups of six animals each. All four-group animals received normal standard diet and water throughout the experimental period which last up to 10 days. Rats were gavaged with vit. E (300 mg/kg b. wt.) daily for 10 days (day 1-10) and were given intraperitoneal injection of 5-FU (150 mg/kg b. wt.) or saline (control) on day 8 to induce mucositis. Results We found that vit. E supplementation ameliorated 5-FU-induced lipid peroxidation, myeloperoxidase activity, activation of nuclear factor κB, expression of cyclooxygenase-2, inducible nitric oxide synthase and mucin depletion. Vit. E administration also attenuated 5-FU-induced histological anomalies such as neutrophil infiltration, loss of cellular integrity, villus and crypt deformities. ConclusionsFindings of the study suggest that vit. E inhibits 5-FU-induced mucositis via modulation of oxidative stress, activation of redox sensitive transcription factor and its downstream targets.

  16. Identifying target regions for vigilance improvement under hormone replacement therapy in postmenopausal syndrome patients by means of electroencephalographic tomography (LORETA).

    PubMed

    Saletu, B; Anderer, P; Saletu-Zyhlarz, G M; Gruber, D; Metka, M; Huber, J

    2005-04-01

    Daytime fatigue, which at the neurophysiological level is due to vigilance decrements, is a frequent complaint in postmenopausal women. In a three-arm, 2-month, parallel group-design study, vigilance-promoting effects of a novel continuous combination (=Climodien 2/3) of estradiol valerate (EV; 2 mg) and dienogest (DNG; 3 mg) were compared with the effects of both EV alone and placebo in 55 insomniac, postmenopausal syndrome patients. Low-resolution brain electromagnetic tomography (LORETA) was undertaken to identify the cerebral target regions of hormone replacement therapy. An omnibus significance test revealed Climodien to increase activity in 882 of 2,394 voxels in the alpha-2 band, followed by 733, 706, and 664 voxels in the beta-2, beta-1, and beta-3 bands, and 509 voxels in the delta band, whereas 2 mg EV alone did not produce a significant suprathreshold activity. Current density increased predominantly in the right hemisphere, which had already been described in the literature as the center of the vigilance system. In the fast alpha range, which plays a major role in the context of vigilance, increased activity was found in the right prefrontal, temporal, and superior parietal cortices, i.e., those brain areas of the right-sided fronto-parietal neuronal network that are responsible for sustained attention. A further activity increase was seen in the anterior cingulate gyrus associated with attentional control and conflict monitoring. The right temporal lobe showed increased current density in all frequency bands. Electroencephalographic tomography (LORETA) identified the right-hemispheric vigilance system as the target region of Climodien.

  17. Tracking target objects orbiting earth using satellite-based telescopes

    DOEpatents

    De Vries, Willem H; Olivier, Scot S; Pertica, Alexander J

    2014-10-14

    A system for tracking objects that are in earth orbit via a constellation or network of satellites having imaging devices is provided. An object tracking system includes a ground controller and, for each satellite in the constellation, an onboard controller. The ground controller receives ephemeris information for a target object and directs that ephemeris information be transmitted to the satellites. Each onboard controller receives ephemeris information for a target object, collects images of the target object based on the expected location of the target object at an expected time, identifies actual locations of the target object from the collected images, and identifies a next expected location at a next expected time based on the identified actual locations of the target object. The onboard controller processes the collected image to identify the actual location of the target object and transmits the actual location information to the ground controller.

  18. Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes

    PubMed Central

    Phamduy, Theresa B.; Chrisey, Douglas B.

    2017-01-01

    Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds’ effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype. PMID:28771473

  19. Identifying High Potential Well Targets with 3D Seismic and Mineralogy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mellors, R. J.

    2015-10-30

    Seismic reflection the primary tool used in petroleum exploration and production, but use in geothermal exploration is less standard, in part due to cost but also due to the challenges in identifying the highly-permeable zones essential for economic hydrothermal systems [e.g. Louie et al., 2011; Majer, 2003]. Newer technology, such as wireless sensors and low-cost high performance computing, has helped reduce the cost and effort needed to conduct 3D surveys. The second difficulty, identifying permeable zones, has been less tractable so far. Here we report on the use of seismic attributes from a 3D seismic survey to identify and mapmore » permeable zones in a hydrothermal area.« less

  20. Targeted sequencing identifies novel variants involved in autosomal recessive hereditary hearing loss in Qatari families.

    PubMed

    Alkowari, Moza K; Vozzi, Diego; Bhagat, Shruti; Krishnamoorthy, Navaneethakrishnan; Morgan, Anna; Hayder, Yousra; Logendra, Barathy; Najjar, Nehal; Gandin, Ilaria; Gasparini, Paolo; Badii, Ramin; Girotto, Giorgia; Abdulhadi, Khalid

    2017-08-01

    Hereditary hearing loss is characterized by a very high genetic heterogeneity. In the Qatari population the role of GJB2, the worldwide HHL major player, seems to be quite limited compared to Caucasian populations. In this study we analysed 18 Qatari families affected by non-syndromic hearing loss using a targeted sequencing approach that allowed us to analyse 81 genes simultaneously. Thanks to this approach, 50% of these families (9 out of 18) resulted positive for the presence of likely causative alleles in 6 different genes: CDH23, MYO6, GJB6, OTOF, TMC1 and OTOA. In particular, 4 novel alleles were detected while the remaining ones were already described to be associated to HHL in other ethnic groups. Molecular modelling has been used to further investigate the role of novel alleles identified in CDH23 and TMC1 genes demonstrating their crucial role in Ca2+ binding and therefore possible functional role in proteins. Present study showed that an accurate molecular diagnosis based on next generation sequencing technologies might largely improve molecular diagnostics outcome leading to benefits for both genetic counseling and definition of recurrence risk. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. A case of myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)-associated hypertrophic pachymeningitis presenting with multiple cranial nerve palsies and diabetes insipidus.

    PubMed

    Yasuda, Ken; Sainouchi, Makoto; Goto, Masahiro; Murase, Nagako; Ohtani, Ryo; Nakamura, Michikazu

    2016-05-31

    A 61-year-old woman developed hearing difficulties and became thirsty after experiencing cold symptoms. A neurological examination revealed a loss of odor sensation, facial palsy, dysphasia, and dysarthria. Vocal cord palsy was observed during pharyngoscopy. Brain magnetic resonance imaging (MRI) showed a thickened pituitary stalk and swelling of the pituitary gland, but no high signal intensity regions were seen in the posterior portion of the pituitary gland. Gadolinium-enhanced MRI demonstrated a thickened dura mater over the anterior cranial fossa. A biopsy specimen of the thickened dura mater showed fibrosis, granulomatous inflammation, and necrotic foci. Blood tests detected myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA). The patient's urine osmolarity was low even though she exhibited hypernatremia. We diagnosed her with hypertrophic pachymeningitis associated with MPO-ANCA and diabetes insipidus. The patient received two courses of 5-day high-dose intravenous methylprednisolone (1.0 g/day), and was subsequently administered oral prednisolone, which gradually relieved her symptoms. However, the patient's symptoms recurred despite the high-dose prednisolone treatment. It was difficult to control the patient's symptoms in this case with oral prednisolone monotherapy, but combined treatment with cyclosporine resulted in sustained remission. It is considered that patients with MPO-ANCA-positive hypertrophic pachymeningitis require combination therapy with prednisolone and immunosuppressive agents at an early stage.

  2. Hospital to Post-Acute Care Facility Transfers: Identifying Targets for Information Exchange Quality Improvement.

    PubMed

    Jones, Christine D; Cumbler, Ethan; Honigman, Benjamin; Burke, Robert E; Boxer, Rebecca S; Levy, Cari; Coleman, Eric A; Wald, Heidi L

    2017-01-01

    Information exchange is critical to high-quality care transitions from hospitals to post-acute care (PAC) facilities. We conducted a survey to evaluate the completeness and timeliness of information transfer and communication between a tertiary-care academic hospital and its related PAC facilities. This was a cross-sectional Web-based 36-question survey of 110 PAC clinicians and staff representing 31 PAC facilities conducted between October and December 2013. We received responses from 71 of 110 individuals representing 29 of 31 facilities (65% and 94% response rates). We collapsed 4-point Likert responses into dichotomous variables to reflect completeness (sufficient vs insufficient) and timeliness (timely vs not timely) for information transfer and communication. Among respondents, 32% reported insufficient information about discharge medical conditions and management plan, and 83% reported at least occasionally encountering problems directly related to inadequate information from the hospital. Hospital clinician contact information was the most common insufficient domain. With respect to timeliness, 86% of respondents desired receipt of a discharge summary on or before the day of discharge, but only 58% reported receiving the summary within this time frame. Through free-text responses, several participants expressed the need for paper prescriptions for controlled pain medications to be sent with patients at the time of transfer. Staff and clinicians at PAC facilities perceive substantial deficits in content and timeliness of information exchange between the hospital and facilities. Such deficits are particularly relevant in the context of the increasing prevalence of bundled payments for care across settings as well as forthcoming readmissions penalties for PAC facilities. Targets identified for quality improvement include structuring discharge summary information to include information identified as deficient by respondents, completion of discharge summaries

  3. Targeted exome sequencing identifies novel compound heterozygous mutations in P3H1 in a fetus with osteogenesis imperfecta type VIII.

    PubMed

    Huang, Yanru; Mei, Libin; Lv, Weigang; Li, Haoxian; Zhang, Rui; Pan, Qian; Tan, Hu; Guo, Jing; Luo, Xiaomei; Chen, Chen; Liang, Desheng; Wu, Lingqian

    2017-01-01

    Osteogenesis imperfecta (OI) is a highly clinically and genetically heterogeneous group of disorders. It is difficult to identify severe OI in the perinatal period. Here, a Chinese woman with a suspected history of fetal OI was referred to our institution at 19weeks of gestation, due to ultrasound inspection during antenatal screening, which revealed bulbous metaphyses, short humeri, and short thick bent femora in the fetus. Using targeted exome sequencing of 248 genes known to be involved in skeletal system diseases, we identified novel compound heterozygous mutation in the P3H1 gene in the fetus with OI type VIII: c.105_120del (p.D36Rfs*16) and c.2164C>T (p.Q722*). These two mutations were inherited from the father and mother, respectively. The mRNA level of P3H1 wasn't changed suggested that mRNA with this mutation escaped from nonsense-mediated RNA decay. Besides, the level of P3H1 was absence while the CRTAP was mildly decreased. In conclusion, our findings imply this novel compound heterozygous mutation as the molecular pathogenetic in a Chinese fetus with OI type VIII, and demonstrate that targeted next-generation sequencing (NGS) is an accurate, rapid, and cost-effective method in the genetic diagnosis of fetal skeletal dysplasia with genetic and clinical heterogeneity, especially for autosomal recessive skeletal disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Target enrichment and high-throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond-Blackfan anaemia.

    PubMed

    Gerrard, Gareth; Valgañón, Mikel; Foong, Hui En; Kasperaviciute, Dalia; Iskander, Deena; Game, Laurence; Müller, Michael; Aitman, Timothy J; Roberts, Irene; de la Fuente, Josu; Foroni, Letizia; Karadimitris, Anastasios

    2013-08-01

    Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA. © 2013 John Wiley & Sons Ltd.

  5. Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3.

    PubMed

    Eisenberger, Tobias; Slim, Rima; Mansour, Ahmad; Nauck, Markus; Nürnberg, Gudrun; Nürnberg, Peter; Decker, Christian; Dafinger, Claudia; Ebermann, Inga; Bergmann, Carsten; Bolz, Hanno Jörn

    2012-09-02

    Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family. Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype. Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis.

  6. Identifying transposon insertions and their effects from RNA-sequencing data.

    PubMed

    de Ruiter, Julian R; Kas, Sjors M; Schut, Eva; Adams, David J; Koudijs, Marco J; Wessels, Lodewyk F A; Jonkers, Jos

    2017-07-07

    Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and novel therapeutic target in acute myeloid leukemia

    PubMed Central

    Porter, Christopher C.; Kim, Jihye; Fosmire, Susan; Gearheart, Christy M.; van Linden, Annemie; Baturin, Dmitry; Zaberezhnyy, Vadym; Patel, Purvi R.; Gao, Dexiang; Tan, Aik Choon; DeGregori, James

    2011-01-01

    Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide shRNA screens to identify proteins that mediate AML cell fate after cytarabine exposure, gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context, and examination of existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. PMID:22289989

  8. Novel high throughput pooled shRNA screening identifies NQO1 as a potential drug target for host directed therapy for tuberculosis

    PubMed Central

    Li, Qing; Karim, Ahmad F.; Ding, Xuedong; Das, Biswajit; Dobrowolski, Curtis; Gibson, Richard M.; Quiñones-Mateu, Miguel E.; Karn, Jonathan; Rojas, Roxana E.

    2016-01-01

    Chemical regulation of macrophage function is one key strategy for developing host-directed adjuvant therapies for tuberculosis (TB). A critical step to develop these therapies is the identification and characterization of specific macrophage molecules and pathways with a high potential to serve as drug targets. Using a barcoded lentivirus-based pooled short-hairpin RNA (shRNA) library combined with next generation sequencing, we identified 205 silenced host genes highly enriched in mycobacteria-resistant macrophages. Twenty-one of these “hits” belonged to the oxidoreductase functional category. NAD(P)H:quinone oxidoreductase 1 (NQO1) was the top oxidoreductase “hit”. NQO1 expression was increased after mycobacterial infection, and NQO1 knockdown increased macrophage differentiation, NF-κB activation, and the secretion of pro-inflammatory cytokines TNF-α and IL-1β in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and anti-bacterial functions. The competitive inhibitor of NQO1 dicoumarol synergized with rifampin to promote intracellular killing of mycobacteria. Thus, NQO1 is a new host target in mycobacterial infection that could potentially be exploited to increase antibiotic efficacy in vivo. Our findings also suggest that pooled shRNA libraries could be valuable tools for genome-wide screening in the search for novel druggable host targets for adjunctive TB therapies. PMID:27297123

  9. Myeloperoxidase Oxidized LDL Interferes with Endothelial Cell Motility through miR-22 and Heme Oxygenase 1 Induction: Possible Involvement in Reendothelialization of Vascular Injuries

    PubMed Central

    Daher, Jalil; Martin, Maud; Rousseau, Alexandre; Nuyens, Vincent; Fayyad-Kazan, Hussein; Van Antwerpen, Pierre; Courbebaisse, Guy; Martiat, Philippe; Badran, Bassam; Dequiedt, Frank

    2014-01-01

    Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque. PMID:25530680

  10. Identifying and targeting cancer stem cells in the treatment of gastric cancer.

    PubMed

    Bekaii-Saab, Tanios; El-Rayes, Bassel

    2017-04-15

    Current treatment regimens for gastric cancer are not adequate. Cancer stem cells (CSCs) may be a key driving factor for growth and metastasis of this tumor type. In contrast to the conventional clonal evolution hypothesis, CSCs can initiate tumor formation, self-renew, and differentiate into tumor-propagating cells. Because gastric cancer can originate from CSCs, it is necessary to review current targets of signaling pathways for CSCs in gastric cancer that are being studied in clinical trials. These pathways are known to regulate the self-renewal and differentiation process in gastric CSCs. A better understanding of the clinical results of trials that target gastric CSCs will lead to better outcomes for patients with gastric cancer. Cancer 2017;123:1303-1312. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

  11. A variant in myeloperoxidase promoter hastens the emergence of hepatocellular carcinoma in patients with HCV-related cirrhosis.

    PubMed

    Nahon, Pierre; Sutton, Angela; Rufat, Pierre; Charnaux, Nathalie; Mansouri, Abdellah; Moreau, Richard; Ganne-Carrié, Nathalie; Grando-Lemaire, Véronique; N'Kontchou, Gisèle; Trinchet, Jean-Claude; Pessayre, Dominique; Beaugrand, Michel

    2012-02-01

    Genetic dimorphisms modulate the activities of several pro- or antioxidant enzymes, including myeloperoxidase (MPO), catalase (CAT), manganese superoxide dismutase (SOD2), and glutathione peroxidase 1 (GPx1). We assessed the role of the G(-463)A-MPO, T(-262)C-CAT, Ala16Val-SOD2, and Pro198Leu-GPx1 variants in modulating HCC development in patients with HCV-induced cirrhosis. Two hundred and five patients with HCV-induced, biopsy-proven cirrhosis but without detectable HCC at inclusion were prospectively followed-up for HCC development. The influence of various genotypes on HCC occurrence was assessed with the Kaplan-Meier method. During follow-up (103.2±3.4 months), 84 patients (41%) developed HCC, and 66 died. Whereas the Ala16Val-SOD2 or Pro198Leu-GPx1 dimorphisms did not modulate the risk, HCC occurrence was increased in patients with either the homozygous GG-MPO genotype (HR=2.8 [1.7-4.4]; first quartile time to HCC occurrence: 45 vs. 96 months; LogRank <0.0001) or the homozygous CC-CAT genotype (HR=1.74 [1.06-2.82]; first quartile time to HCC occurrence: 55 vs. 96 months; LogRank=0.02). Compared to patients with neither of these two at risk factors, patients with only the CC-CAT genotype had a HR of 2.05 [0.9-4.6] (p=0.08) and patients with only the GG-MPO genotype had a HR of 3.8 [1.5-9.1] (p=0.002), while patients with both risk factors had an HR of 4.8 [2.2-10.4] (p<0.0001). However, only the GG-MPO genotype was independently associated with the HCC risk in multivariate Cox analysis. The high activity-associated GG-MPO genotype increases the rate of HCC occurrence in patients with HCV-induced cirrhosis. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  12. Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

    PubMed

    Angeloni, Cristina; Turroni, Silvia; Bianchi, Laura; Fabbri, Daniele; Motori, Elisa; Malaguti, Marco; Leoncini, Emanuela; Maraldi, Tullia; Bini, Luca; Brigidi, Patrizia; Hrelia, Silvana

    2013-01-01

    Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

  13. Comprehensive metabolomic, lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis

    DOE PAGES

    Pomraning, Kyle R.; Wei, Siwei; Karagiosis, Sue A.; ...

    2015-04-23

    Yarrowia lipolytica is an oleaginous ascomycete yeast that accumulates large amounts of lipids and has potential as a biofuel producing organism. Despite a growing scientific literature focused on lipid production by Y. lipolytica, there remain significant knowledge gaps regarding the key biological processes involved. We applied a combination of metabolomic and lipidomic profiling approaches as well as microscopic techniques to identify and characterize the key pathways involved in de novo lipid accumulation from glucose in batch cultured, wild-type Y. lipolytica. We found that lipids accumulated rapidly and peaked at 48 hours during the five day experiment, concurrent with a shiftmore » in amino acid metabolism. We also report that Y. lipolytica secretes disaccharides early in batch culture and reabsorbs them when extracellular glucose is depleted. Exhaustion of extracellular sugars coincided with thickening of the cell wall, suggesting that genes involved in cell wall biogenesis may be a useful target for improving the efficiency of lipid producing yeast strains.« less

  14. Integration of targeted metabolomics and transcriptomics identifies deregulation of phosphatidylcholine metabolism in Huntington's disease peripheral blood samples.

    PubMed

    Mastrokolias, Anastasios; Pool, Rene; Mina, Eleni; Hettne, Kristina M; van Duijn, Erik; van der Mast, Roos C; van Ommen, GertJan; 't Hoen, Peter A C; Prehn, Cornelia; Adamski, Jerzy; van Roon-Mom, Willeke

    Metabolic changes have been frequently associated with Huntington's disease (HD). At the same time peripheral blood represents a minimally invasive sampling avenue with little distress to Huntington's disease patients especially when brain or other tissue samples are difficult to collect. We investigated the levels of 163 metabolites in HD patient and control serum samples in order to identify disease related changes. Additionally, we integrated the metabolomics data with our previously published next generation sequencing-based gene expression data from the same patients in order to interconnect the metabolomics changes with transcriptional alterations. This analysis was performed using targeted metabolomics and flow injection electrospray ionization tandem mass spectrometry in 133 serum samples from 97 Huntington's disease patients (29 pre-symptomatic and 68 symptomatic) and 36 controls. By comparing HD mutation carriers with controls we identified 3 metabolites significantly changed in HD (serine and threonine and one phosphatidylcholine-PC ae C36:0) and an additional 8 phosphatidylcholines (PC aa C38:6, PC aa C36:0, PC ae C38:0, PC aa C38:0, PC ae C38:6, PC ae C42:0, PC aa C36:5 and PC ae C36:0) that exhibited a significant association with disease severity. Using workflow based exploitation of pathway databases and by integrating our metabolomics data with our gene expression data from the same patients we identified 4 deregulated phosphatidylcholine metabolism related genes ( ALDH1B1 , MBOAT1 , MTRR and PLB1 ) that showed significant association with the changes in metabolite concentrations. Our results support the notion that phosphatidylcholine metabolism is deregulated in HD blood and that these metabolite alterations are associated with specific gene expression changes.

  15. Targeted resequencing identifies defective variants of decoy receptor 3 in pediatric-onset inflammatory bowel disease.

    PubMed

    Cardinale, C J; Wei, Z; Panossian, S; Wang, F; Kim, C E; Mentch, F D; Chiavacci, R M; Kachelries, K E; Pandey, R; Grant, S F A; Baldassano, R N; Hakonarson, H

    2013-10-01

    Genome-wide association studies have implicated common variation at the 20q13 locus in inflammatory bowel disease, particularly for the pediatric Crohn's form. This locus harbors tumor necrosis factor receptor superfamily (TNFRSF6B), encoding a secreted protein, decoy receptor 3 (DcR3), which binds to and neutralizes pro-inflammatory cytokines of the tumor necrosis factor superfamily. We sought to further the evidence of DcR3's role in pediatric IBD by identifying missense mutations with functional significance within TNFRSF6B. We sequenced the exons of the gene in 528 Caucasian pediatric IBD cases and 549 Caucasian healthy controls to establish the frequency of such events in each population. Sequencing revealed that our IBD cohort harbored a greater number of missense variants, yielding an odds ratio of 3.9 (P-value=0.005). Using functional assays, we established that the frequency of mutants defective in secretion from cultured cells was greater in the Crohn's category than in the controls, yielding an odds ratio of 7.1 (P-value=0.004). These results suggest that rare defective variants in TNFRSF6B have a role in the pathogenesis of some cases of IBD and that interventions targeting this group of tumor necrosis factor-family members may benefit patients with IBD.

  16. Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate*

    PubMed Central

    Schwarz, Jennifer Jasmin; Wiese, Heike; Tölle, Regine Charlotte; Zarei, Mostafa; Dengjel, Jörn; Warscheid, Bettina; Thedieck, Kathrin

    2015-01-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation. PMID:25907765

  17. Neutrophil Collagenase, Gelatinase and Myeloperoxidase in Tears of Stevens-Johnson Syndrome and Ocular Cicatricial Pemphigoid Patients

    PubMed Central

    Arafat, Samer N.; Suelves, Ana M.; Spurr-Michaud, Sandra; Chodosh, James; Foster, C. Stephen; Dohlman, Claes H.; Gipson, Ilene K.

    2013-01-01

    Objective To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP). Design Prospective non-interventional cohort study. Participants Four SJS patients (7 eyes), 19 OCP patients (37 eyes) and 20 post-phacoemulsification healthy controls (40 eyes). Methods Tear washes were collected from all patients and were analyzed for levels of MMP-2, -3, -7, -8, -9, -12, MPO and TIMP-1 using multi-analyte bead-based enzyme-linked immunosorbent assays (ELISA). Total MMP activity was determined using a fluorimetric assay. Correlation studies were performed between the various analytes within study groups. Main Outcome Measures Levels of MMP-2, -3, -7, -8, -9, -12, MPO and TIMP-1 (in ng/µg protein), total MMP activity (in relative fluorescent units/min/µg protein) in tears, MMP-8/TIMP-1, MMP-9/TIMP-1 ratios and the correlations between MMP-8 and MMP-9 and each MMP and MPO. Results MMP-8, MMP-9 and MPO levels were significantly elevated in SJS and OCP tears (SJS > OCP) when compared to controls. MMP activity was highest in SJS while OCP and controls showed lower and similar activities. TIMP-1 levels were decreased in SJS and OCP when compared to controls with OCP levels reaching significance. MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS > OCP) when compared to controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels and MMP-8 correlated with MPO but did not reach significance in SJS. There was no relationship between MMP-7 and MPO. Conclusions Since MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes including MMP-9 in SJS and OCP tears. Elevated MMP/TIMP ratios and MMP activity suggest an imbalance in tear MMP regulation

  18. Targeting hardwoods

    Treesearch

    Douglass F. Jacobs

    2011-01-01

    Increasing demand for hardwood seedlings has prompted research to identify target seedling characteristics that promote hardwood plantation establishment. Operational establishment of hardwood plantations has typically emphasized seed collection from non-improved genetic sources, bareroot nursery seedling production, and spring planting using machine planters. The...

  19. Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3

    PubMed Central

    2012-01-01

    Background Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family. Methods Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. Results Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype. Conclusion Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis. PMID:22938382

  20. Statistical methods for identifying and bounding a UXO target area or minefield

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McKinstry, Craig A.; Pulsipher, Brent A.; Gilbert, Richard O.

    2003-09-18

    The sampling unit for minefield or UXO area characterization is typically represented by a geographical block or transect swath that lends itself to characterization by geophysical instrumentation such as mobile sensor arrays. New spatially based statistical survey methods and tools, more appropriate for these unique sampling units have been developed and implemented at PNNL (Visual Sample Plan software, ver. 2.0) with support from the US Department of Defense. Though originally developed to support UXO detection and removal efforts, these tools may also be used in current form or adapted to support demining efforts and aid in the development of newmore » sensors and detection technologies by explicitly incorporating both sampling and detection error in performance assessments. These tools may be used to (1) determine transect designs for detecting and bounding target areas of critical size, shape, and density of detectable items of interest with a specified confidence probability, (2) evaluate the probability that target areas of a specified size, shape and density have not been missed by a systematic or meandering transect survey, and (3) support post-removal verification by calculating the number of transects required to achieve a specified confidence probability that no UXO or mines have been missed.« less

  1. TARGET Research Goals

    Cancer.gov

    TARGET researchers use various sequencing and array-based methods to examine the genomes, transcriptomes, and for some diseases epigenomes of select childhood cancers. This “multi-omic” approach generates a comprehensive profile of molecular alterations for each cancer type. Alterations are changes in DNA or RNA, such as rearrangements in chromosome structure or variations in gene expression, respectively. Through computational analyses and assays to validate biological function, TARGET researchers predict which alterations disrupt the function of a gene or pathway and promote cancer growth, progression, and/or survival. Researchers identify candidate therapeutic targets and/or prognostic markers from the cancer-associated alterations.

  2. TargetCompare: A web interface to compare simultaneous miRNAs targets.

    PubMed

    Moreira, Fabiano Cordeiro; Dustan, Bruno; Hamoy, Igor G; Ribeiro-Dos-Santos, André M; Dos Santos, Andrea Ribeiro

    2014-01-01

    MicroRNAs (miRNAs) are small non-coding nucleotide sequences between 17 and 25 nucleotides in length that primarily function in the regulation of gene expression. A since miRNA has thousand of predict targets in a complex, regulatory cell signaling network. Therefore, it is of interest to study multiple target genes simultaneously. Hence, we describe a web tool (developed using Java programming language and MySQL database server) to analyse multiple targets of pre-selected miRNAs. We cross validated the tool in eight most highly expressed miRNAs in the antrum region of stomach. This helped to identify 43 potential genes that are target of at least six of the referred miRNAs. The developed tool aims to reduce the randomness and increase the chance of selecting strong candidate target genes and miRNAs responsible for playing important roles in the studied tissue. http://lghm.ufpa.br/targetcompare.

  3. Integrative analysis identifies targetable CREB1/FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence.

    PubMed

    Sunkel, Benjamin; Wu, Dayong; Chen, Zhong; Wang, Chiou-Miin; Liu, Xiangtao; Ye, Zhenqing; Horning, Aaron M; Liu, Joseph; Mahalingam, Devalingam; Lopez-Nicora, Horacio; Lin, Chun-Lin; Goodfellow, Paul J; Clinton, Steven K; Jin, Victor X; Chen, Chun-Liang; Huang, Tim H-M; Wang, Qianben

    2016-05-19

    Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Identifying potential RNAi targets in grain aphid (Sitobion avenae F.) based on transcriptome profiling of its alimentary canal after feeding on wheat plants.

    PubMed

    Zhang, Min; Zhou, Yuwen; Wang, Hui; Jones, Huw; Gao, Qiang; Wang, Dahai; Ma, Youzhi; Xia, Lanqin

    2013-08-16

    The grain aphid (Sitobion avenae F.) is a major agricultural pest which causes significant yield losses of wheat in China, Europe and North America annually. Transcriptome profiling of the grain aphid alimentary canal after feeding on wheat plants could provide comprehensive gene expression information involved in feeding, ingestion and digestion. Furthermore, selection of aphid-specific RNAi target genes would be essential for utilizing a plant-mediated RNAi strategy to control aphids via a non-toxic mode of action. However, due to the tiny size of the alimentary canal and lack of genomic information on grain aphid as a whole, selection of the RNAi targets is a challenging task that as far as we are aware, has never been documented previously. In this study, we performed de novo transcriptome assembly and gene expression analyses of the alimentary canals of grain aphids before and after feeding on wheat plants using Illumina RNA sequencing. The transcriptome profiling generated 30,427 unigenes with an average length of 664 bp. Furthermore, comparison of the transcriptomes of alimentary canals of pre- and post feeding grain aphids indicated that 5490 unigenes were differentially expressed, among which, diverse genes and/or pathways were identified and annotated. Based on the RPKM values of these unigenes, 16 of them that were significantly up or down-regulated upon feeding were selected for dsRNA artificial feeding assay. Of these, 5 unigenes led to higher mortality and developmental stunting in an artificial feeding assay due to the down-regulation of the target gene expression. Finally, by adding fluorescently labelled dsRNA into the artificial diet, the spread of fluorescence signal in the whole body tissues of grain aphid was observed. Comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphids on wheat plants provided comprehensive gene expression information that could facilitate our understanding of the molecular

  5. Identifying potential RNAi targets in grain aphid (Sitobion avenae F.) based on transcriptome profiling of its alimentary canal after feeding on wheat plants

    PubMed Central

    2013-01-01

    Background The grain aphid (Sitobion avenae F.) is a major agricultural pest which causes significant yield losses of wheat in China, Europe and North America annually. Transcriptome profiling of the grain aphid alimentary canal after feeding on wheat plants could provide comprehensive gene expression information involved in feeding, ingestion and digestion. Furthermore, selection of aphid-specific RNAi target genes would be essential for utilizing a plant-mediated RNAi strategy to control aphids via a non-toxic mode of action. However, due to the tiny size of the alimentary canal and lack of genomic information on grain aphid as a whole, selection of the RNAi targets is a challenging task that as far as we are aware, has never been documented previously. Results In this study, we performed de novo transcriptome assembly and gene expression analyses of the alimentary canals of grain aphids before and after feeding on wheat plants using Illumina RNA sequencing. The transcriptome profiling generated 30,427 unigenes with an average length of 664 bp. Furthermore, comparison of the transcriptomes of alimentary canals of pre- and post feeding grain aphids indicated that 5490 unigenes were differentially expressed, among which, diverse genes and/or pathways were identified and annotated. Based on the RPKM values of these unigenes, 16 of them that were significantly up or down-regulated upon feeding were selected for dsRNA artificial feeding assay. Of these, 5 unigenes led to higher mortality and developmental stunting in an artificial feeding assay due to the down-regulation of the target gene expression. Finally, by adding fluorescently labelled dsRNA into the artificial diet, the spread of fluorescence signal in the whole body tissues of grain aphid was observed. Conclusions Comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphids on wheat plants provided comprehensive gene expression information that could facilitate our

  6. Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

    PubMed Central

    Masuno, Kiriko; Haldar, Saptarsi M.; Jeyaraj, Darwin; Mailloux, Christina M.; Huang, Xiaozhu; Panettieri, Rey A.; Jain, Mukesh K.

    2011-01-01

    Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function. PMID:21257922

  7. Candidiasis drug discovery and development: new approaches targeting virulence for discovering and identifying new drugs

    PubMed Central

    Pierce, Christopher G.; Lopez-Ribot, Jose L.

    2014-01-01

    Introduction Targeting pathogenetic mechanisms rather than essential processes represents a very attractive alternative for the development of new antibiotics. This may be particularly important in the case of antimycotics, due to the urgent need for novel antifungal drugs and the paucity of selective fungal targets. The opportunistic pathogenic fungus Candida albicans is the main etiological agent of candidiasis, the most common human fungal infection. These infections carry unacceptably high mortality rates, a clear reflection of the many shortcomings of current antifungal therapy, including the limited armamentarium of antifungal agents, their toxicity, and the emergence of resistance. Moreover the antifungal pipeline is mostly dry. Areas covered This review covers some of the most recent progress towards understanding C. albicans pathogenetic processes and how to harness this information for the development of anti-virulence agents. The two principal areas covered are filamentation and biofilm formation, as C. albicans pathogenicity is intimately linked to its ability to undergo morphogenetic conversions between yeast and filamentous morphologies and to its ability to form biofilms. Expert opinion We argue that filamentation and biofilm formation represent high value targets, yet clinically unexploited, for the development of novel anti-virulence approaches against candidiasis. Although this has proved a difficult task despite increasing understanding at the molecular level of C. albicans virulence, we highlight new opportunities and prospects for antifungal drug development targeting these two important biological processes. PMID:23738751

  8. Identifying and Targeting Mortality Disparities: A Framework for Sub-Saharan Africa Using Adult Mortality Data from South Africa

    PubMed Central

    Sartorius, Benn; Sartorius, Kurt

    2013-01-01

    Background Health inequities in developing countries are difficult to eradicate because of limited resources. The neglect of adult mortality in Sub-Saharan Africa (SSA) is a particular concern. Advances in data availability, software and analytic methods have created opportunities to address this challenge and tailor interventions to small areas. This study demonstrates how a generic framework can be applied to guide policy interventions to reduce adult mortality in high risk areas. The framework, therefore, incorporates the spatial clustering of adult mortality, estimates the impact of a range of determinants and quantifies the impact of their removal to ensure optimal returns on scarce resources. Methods Data from a national cross-sectional survey in 2007 were used to illustrate the use of the generic framework for SSA and elsewhere. Adult mortality proportions were analyzed at four administrative levels and spatial analyses were used to identify areas with significant excess mortality. An ecological approach was then used to assess the relationship between mortality “hotspots” and various determinants. Population attributable fractions were calculated to quantify the reduction in mortality as a result of targeted removal of high-impact determinants. Results Overall adult mortality rate was 145 per 10,000. Spatial disaggregation identified a highly non-random pattern and 67 significant high risk local municipalities were identified. The most prominent determinants of adult mortality included HIV antenatal sero-prevalence, low SES and lack of formal marital union status. The removal of the most attributable factors, based on local area prevalence, suggest that overall adult mortality could be potentially reduced by ∼90 deaths per 10,000. Conclusions The innovative use of secondary data and advanced epidemiological techniques can be combined in a generic framework to identify and map mortality to the lowest administration level. The identification of high

  9. Systems genetics for drug target discovery

    PubMed Central

    Penrod, Nadia M.; Cowper-Sal_lari, Richard; Moore, Jason H.

    2011-01-01

    The collection and analysis of genomic data has the potential to reveal novel druggable targets by providing insight into the genetic basis of disease. However, the number of drugs, targeting new molecular entities, approved by the US Food and Drug Administration (FDA) has not increased in the years since the collection of genomic data has become commonplace. The paucity of translatable results can be partly attributed to conventional analysis methods that test one gene at a time in an effort to identify disease-associated factors as candidate drug targets. By disengaging genetic factors from their position within the genetic regulatory system, much of the information stored within the genomic data set is lost. Here we discuss how genomic data is used to identify disease-associated genes or genomic regions, how disease-associated regions are validated as functional targets, and the role network analysis can play in bridging the gap between data generation and effective drug target identification. PMID:21862141

  10. Petri net siphon analysis and graph theoretic measures for identifying combination therapies in cancer.

    PubMed

    Behinaein, Behnam; Rudie, Karen; Sangrar, Waheed

    2016-10-03

    Epidermal Growth Factor Receptor (EGFR) signaling to the Ras-MAPK pathway is implicated in the development and progression of cancer and is a major focus of targeted combination therapies. Physiochemical models have been used for identifying and testing the signal-inhibiting potential of targeted therapies, however, their application to larger multi-pathway networks is limited by the availability of experimentally-determined rate and concentration parameters. An alternate strategy for identifying and evaluating drug-targetable nodes is proposed. A physiochemical model of EGFR-Ras-MAPK signaling is implemented and calibrated to experimental data. Essential topological features of the model are converted into a Petri net and nodes that behave as siphons-a structural property of Petri nets-are identified. Siphons represent potential drug-targets since they are unrecoverable if their values fall below a threshold. Centrality measures are then used to prioritize siphons identified as candidate drug-targets. Single and multiple drug-target combinations are identified which correspond to clinically relevant drug targets and exhibit inhibition synergy in physiochemical simulations of EGF-induced EGFR-Ras-MAPK signaling. Taken together, these studies suggest that siphons and centrality analyses are a promising computational strategy to identify and rank drug-targetable nodes in larger networks as they do not require knowledge of the dynamics of the system, but rely solely on topology.

  11. MicroRNA-Target Network Inference and Local Network Enrichment Analysis Identify Two microRNA Clusters with Distinct Functions in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Sass, Steffen; Pitea, Adriana; Unger, Kristian; Hess, Julia; Mueller, Nikola S.; Theis, Fabian J.

    2015-01-01

    MicroRNAs represent ~22 nt long endogenous small RNA molecules that have been experimentally shown to regulate gene expression post-transcriptionally. One main interest in miRNA research is the investigation of their functional roles, which can typically be accomplished by identification of mi-/mRNA interactions and functional annotation of target gene sets. We here present a novel method “miRlastic”, which infers miRNA-target interactions using transcriptomic data as well as prior knowledge and performs functional annotation of target genes by exploiting the local structure of the inferred network. For the network inference, we applied linear regression modeling with elastic net regularization on matched microRNA and messenger RNA expression profiling data to perform feature selection on prior knowledge from sequence-based target prediction resources. The novelty of miRlastic inference originates in predicting data-driven intra-transcriptome regulatory relationships through feature selection. With synthetic data, we showed that miRlastic outperformed commonly used methods and was suitable even for low sample sizes. To gain insight into the functional role of miRNAs and to determine joint functional properties of miRNA clusters, we introduced a local enrichment analysis procedure. The principle of this procedure lies in identifying regions of high functional similarity by evaluating the shortest paths between genes in the network. We can finally assign functional roles to the miRNAs by taking their regulatory relationships into account. We thoroughly evaluated miRlastic on a cohort of head and neck cancer (HNSCC) patients provided by The Cancer Genome Atlas. We inferred an mi-/mRNA regulatory network for human papilloma virus (HPV)-associated miRNAs in HNSCC. The resulting network best enriched for experimentally validated miRNA-target interaction, when compared to common methods. Finally, the local enrichment step identified two functional clusters of mi

  12. MicroRNA-Target Network Inference and Local Network Enrichment Analysis Identify Two microRNA Clusters with Distinct Functions in Head and Neck Squamous Cell Carcinoma.

    PubMed

    Sass, Steffen; Pitea, Adriana; Unger, Kristian; Hess, Julia; Mueller, Nikola S; Theis, Fabian J

    2015-12-18

    MicroRNAs represent ~22 nt long endogenous small RNA molecules that have been experimentally shown to regulate gene expression post-transcriptionally. One main interest in miRNA research is the investigation of their functional roles, which can typically be accomplished by identification of mi-/mRNA interactions and functional annotation of target gene sets. We here present a novel method "miRlastic", which infers miRNA-target interactions using transcriptomic data as well as prior knowledge and performs functional annotation of target genes by exploiting the local structure of the inferred network. For the network inference, we applied linear regression modeling with elastic net regularization on matched microRNA and messenger RNA expression profiling data to perform feature selection on prior knowledge from sequence-based target prediction resources. The novelty of miRlastic inference originates in predicting data-driven intra-transcriptome regulatory relationships through feature selection. With synthetic data, we showed that miRlastic outperformed commonly used methods and was suitable even for low sample sizes. To gain insight into the functional role of miRNAs and to determine joint functional properties of miRNA clusters, we introduced a local enrichment analysis procedure. The principle of this procedure lies in identifying regions of high functional similarity by evaluating the shortest paths between genes in the network. We can finally assign functional roles to the miRNAs by taking their regulatory relationships into account. We thoroughly evaluated miRlastic on a cohort of head and neck cancer (HNSCC) patients provided by The Cancer Genome Atlas. We inferred an mi-/mRNA regulatory network for human papilloma virus (HPV)-associated miRNAs in HNSCC. The resulting network best enriched for experimentally validated miRNA-target interaction, when compared to common methods. Finally, the local enrichment step identified two functional clusters of miRNAs that

  13. ARID1B alterations identify aggressive tumors in neuroblastoma.

    PubMed

    Lee, Soo Hyun; Kim, Jung-Sun; Zheng, Siyuan; Huse, Jason T; Bae, Joon Seol; Lee, Ji Won; Yoo, Keon Hee; Koo, Hong Hoe; Kyung, Sungkyu; Park, Woong-Yang; Sung, Ki W

    2017-07-11

    Targeted panel sequencing was performed to determine molecular targets and biomarkers in 72 children with neuroblastoma. Frequent genetic alterations were detected in ALK (16.7%), BRCA1 (13.9%), ATM (12.5%), and PTCH1 (11.1%) in an 83-gene panel. Molecular targets for targeted therapy were identified in 16 of 72 patients (22.2%). Two-thirds of ALK mutations were known to increase sensitivity to ALK inhibitors. Sequence alterations in ARID1B were identified in 5 of 72 patients (6.9%). Four of five ARID1B alterations were detected in tumors of high-risk patients. Two of five patients with ARID1B alterations died of disease progression. Relapse-free survival was lower in patients with ARID1B alterations than in those without (p = 0.01). In analysis confined to high-risk patients, 3-year overall survival was lower in patients with an ARID1B alteration (33.3 ± 27.2%) or MYCN amplification (30.0 ± 23.9%) than in those with neither ARID1B alteration nor MYCN amplification (90.5 ± 6.4%, p = 0.05). These results provide possibilities for targeted therapy and a new biomarker identifying a subgroup of neuroblastoma patients with poor prognosis.

  14. TargetCompare: A web interface to compare simultaneous miRNAs targets

    PubMed Central

    Moreira, Fabiano Cordeiro; Dustan, Bruno; Hamoy, Igor G; Ribeiro-dos-Santos, André M; dos Santos, Ândrea Ribeiro

    2014-01-01

    MicroRNAs (miRNAs) are small non-coding nucleotide sequences between 17 and 25 nucleotides in length that primarily function in the regulation of gene expression. A since miRNA has thousand of predict targets in a complex, regulatory cell signaling network. Therefore, it is of interest to study multiple target genes simultaneously. Hence, we describe a web tool (developed using Java programming language and MySQL database server) to analyse multiple targets of pre-selected miRNAs. We cross validated the tool in eight most highly expressed miRNAs in the antrum region of stomach. This helped to identify 43 potential genes that are target of at least six of the referred miRNAs. The developed tool aims to reduce the randomness and increase the chance of selecting strong candidate target genes and miRNAs responsible for playing important roles in the studied tissue. Availability http://lghm.ufpa.br/targetcompare PMID:25352731

  15. Identifying specific beliefs to target to improve restaurant employees' intentions for performing three important food safety behaviors.

    PubMed

    Pilling, Valerie K; Brannon, Laura A; Shanklin, Carol W; Howells, Amber D; Roberts, Kevin R

    2008-06-01

    Current national food safety training programs appear ineffective at improving food safety practices in foodservice operations, given the substantial number of Americans affected by foodborne illnesses after eating in restaurants each year. The Theory of Planned Behavior (TpB) was used to identify important beliefs that may be targeted to improve foodservice employees' intentions for three food safety behaviors that have the most substantial affect on public health: hand washing, using thermometers, and proper handling of food contact surfaces. In a cross-sectional design, foodservice employees (n=190) across three midwestern states completed a survey assessing TpB components and knowledge for the three food safety behaviors. Multiple regression analyses were performed on the TpB components for each behavior. Independent-samples t tests identified TpB beliefs that discriminated between participants who absolutely intend to perform the behaviors and those with lower intention. Employees' attitudes were the one consistent predictor of intentions for performing all three behaviors. However, a unique combination of important predictors existed for each separate behavior. Interventions for improving employees' behavioral intentions for food safety should focus on TpB components that predict intentions for each behavior and should bring all employees' beliefs in line with those of the employees who already intend to perform the food safety behaviors. Registered dietitians; dietetic technicians, registered; and foodservice managers can use these results to enhance training sessions and motivational programs to improve employees' food safety behaviors. Results also assist these professionals in recognizing their responsibility for enforcing and providing adequate resources for proper food safety behaviors.

  16. Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin

    PubMed Central

    Hauck, Nastasja C.; Kirpach, Josiane; Kiefer, Christina; Farinelle, Sophie; Morris, Stephen A.; Muller, Claude P.; Lu, I-Na

    2018-01-01

    To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants. PMID:29587397

  17. Identifying Medication Targets for Psychostimulant Addiction: Unraveling the Dopamine D3 Receptor Hypothesis

    PubMed Central

    2016-01-01

    The dopamine D3 receptor (D3R) is a target for developing medications to treat substance use disorders. D3R-selective compounds with high affinity and varying efficacies have been discovered, providing critical research tools for cell-based studies that have been translated to in vivo models of drug abuse. D3R antagonists and partial agonists have shown especially promising results in rodent models of relapse-like behavior, including stress-, drug-, and cue-induced reinstatement of drug seeking. However, to date, translation to human studies has been limited. Herein, we present an overview and illustrate some of the pitfalls and challenges of developing novel D3R-selective compounds toward clinical utility, especially for treatment of cocaine abuse. Future research and development of D3R-selective antagonists and partial agonists for substance abuse remains critically important but will also require further evaluation and development of translational animal models to determine the best time in the addiction cycle to target D3Rs for optimal therapeutic efficacy. PMID:25826710

  18. Cancer in silico drug discovery: a systems biology tool for identifying candidate drugs to target specific molecular tumor subtypes.

    PubMed

    San Lucas, F Anthony; Fowler, Jerry; Chang, Kyle; Kopetz, Scott; Vilar, Eduardo; Scheet, Paul

    2014-12-01

    Large-scale cancer datasets such as The Cancer Genome Atlas (TCGA) allow researchers to profile tumors based on a wide range of clinical and molecular characteristics. Subsequently, TCGA-derived gene expression profiles can be analyzed with the Connectivity Map (CMap) to find candidate drugs to target tumors with specific clinical phenotypes or molecular characteristics. This represents a powerful computational approach for candidate drug identification, but due to the complexity of TCGA and technology differences between CMap and TCGA experiments, such analyses are challenging to conduct and reproduce. We present Cancer in silico Drug Discovery (CiDD; scheet.org/software), a computational drug discovery platform that addresses these challenges. CiDD integrates data from TCGA, CMap, and Cancer Cell Line Encyclopedia (CCLE) to perform computational drug discovery experiments, generating hypotheses for the following three general problems: (i) determining whether specific clinical phenotypes or molecular characteristics are associated with unique gene expression signatures; (ii) finding candidate drugs to repress these expression signatures; and (iii) identifying cell lines that resemble the tumors being studied for subsequent in vitro experiments. The primary input to CiDD is a clinical or molecular characteristic. The output is a biologically annotated list of candidate drugs and a list of cell lines for in vitro experimentation. We applied CiDD to identify candidate drugs to treat colorectal cancers harboring mutations in BRAF. CiDD identified EGFR and proteasome inhibitors, while proposing five cell lines for in vitro testing. CiDD facilitates phenotype-driven, systematic drug discovery based on clinical and molecular data from TCGA. ©2014 American Association for Cancer Research.

  19. Targeted Endoscopic Imaging

    PubMed Central

    Li, Meng; Wang, Thomas D

    2011-01-01

    Summary Endoscopy has undergone explosive technological growth in over recent years, and with the emergence of targeted imaging, its truly transformative power and impact in medicine lies just over the horizon. Today, our ability to see inside the digestive tract with medical endoscopy is headed toward exciting crossroads. The existing paradigm of making diagnostic decisions based on observing structural changes and identifying anatomical landmarks may soon be replaced by visualizing functional properties and imaging molecular expression. In this novel approach, the presence of intracellular and cell surface targets unique to disease are identified and used to predict the likelihood of mucosal transformation and response to therapy. This strategy can result in the development of new methods for early cancer detection, personalized therapy, and chemoprevention. This targeted approach will require further development of molecular probes and endoscopic instruments, and will need support from the FDA for streamlined regulatory oversight. Overall, this molecular imaging modality promises to significantly broaden the capabilities of the gastroenterologist by providing a new approach to visualize the mucosa of the digestive tract in a manner that has never been seen before. PMID:19423025

  20. Developing clinical practice guidelines: target audiences, identifying topics for guidelines, guideline group composition and functioning and conflicts of interest

    PubMed Central

    2012-01-01

    Clinical practice guidelines are one of the foundations of efforts to improve health care. In 1999, we authored a paper about methods to develop guidelines. Since it was published, the methods of guideline development have progressed both in terms of methods and necessary procedures and the context for guideline development has changed with the emergence of guideline clearing houses and large scale guideline production organisations (such as the UK National Institute for Health and Clinical Excellence). It therefore seems timely to, in a series of three articles, update and extend our earlier paper. In this first paper we discuss: the target audience(s) for guidelines and their use of guidelines; identifying topics for guidelines; guideline group composition (including consumer involvement) and the processes by which guideline groups function and the important procedural issue of managing conflicts of interest in guideline development. PMID:22762776

  1. Developing clinical practice guidelines: target audiences, identifying topics for guidelines, guideline group composition and functioning and conflicts of interest.

    PubMed

    Eccles, Martin P; Grimshaw, Jeremy M; Shekelle, Paul; Schünemann, Holger J; Woolf, Steven

    2012-07-04

    Clinical practice guidelines are one of the foundations of efforts to improve health care. In 1999, we authored a paper about methods to develop guidelines. Since it was published, the methods of guideline development have progressed both in terms of methods and necessary procedures and the context for guideline development has changed with the emergence of guideline clearing houses and large scale guideline production organisations (such as the UK National Institute for Health and Clinical Excellence). It therefore seems timely to, in a series of three articles, update and extend our earlier paper. In this first paper we discuss: the target audience(s) for guidelines and their use of guidelines; identifying topics for guidelines; guideline group composition (including consumer involvement) and the processes by which guideline groups function and the important procedural issue of managing conflicts of interest in guideline development.

  2. Associations of periparturient plasma biochemical parameters, endometrial leukocyte esterase and myeloperoxidase, and bacterial detection with clinical and subclinical endometritis in postpartum dairy cows

    PubMed Central

    NAZHAT, Sayed Ahmad; KITAHARA, Go; KOZUKA, Nobutoshi; MIDO, Shogo; SADAWY, Mohammed; ALI, Hossam El-Sheikh; OSAWA, Takeshi

    2018-01-01

    This study was aimed at demonstrating associations between peripheral biochemical parameters, endometrial leukocyte esterase (LE) and myeloperoxidase (MPO), and bacterial detection with the degree of endometrial inflammation, and determining the best time postpartum for diagnosing endometritis to predict subsequent fertility in dairy cows. Plasma albumin, blood urea nitrogen (BUN), total cholesterol (T-cho), NEFA, and BHBA concentrations were analyzed in 43 Holstein cows at 3, 5 and 7 weeks postpartum (W3, W5 and W7). Endometrial samples were collected at W3, W5 and W7 to examine LE and MPO activities, bacterial detection rates, and PMN% profiles. The 43 cows were divided into healthy (HE), subclinical endometritis (SE), and clinical endometritis (CE) groups, classified differently at W3, W5 and W7 based on the definitions of SE and CE for each of the three weeks pp. LE level had an association with PMN% in all weeks pp (P<0.05). Albumin and BUN levels had weak negative associations with endometrial PMN% at W3. Pathogenic bacterial detection rates were higher in the cows with endometritis at W3 and W5. Conception rate at first artificial insemination tended to be lower (P=0.057) in the cows diagnosed with endometritis at W3 than in the healthy cows. In conclusion, associations were found between endometrial LE and endometritis, but not for MPO and endometritis. Diagnosing endometritis in W3 may be the best moment to predict subsequent fertility. PMID:29332864

  3. High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

    PubMed Central

    Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

    2016-01-01

    Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

  4. A tale of two sequences: microRNA-target chimeric reads.

    PubMed

    Broughton, James P; Pasquinelli, Amy E

    2016-04-04

    In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

  5. Measuring Health Information Dissemination and Identifying Target Interest Communities on Twitter: Methods Development and Case Study of the @SafetyMD Network.

    PubMed

    Kandadai, Venk; Yang, Haodong; Jiang, Ling; Yang, Christopher C; Fleisher, Linda; Winston, Flaura Koplin

    2016-05-05

    Little is known about the ability of individual stakeholder groups to achieve health information dissemination goals through Twitter. This study aimed to develop and apply methods for the systematic evaluation and optimization of health information dissemination by stakeholders through Twitter. Tweet content from 1790 followers of @SafetyMD (July-November 2012) was examined. User emphasis, a new indicator of Twitter information dissemination, was defined and applied to retweets across two levels of retweeters originating from @SafetyMD. User interest clusters were identified based on principal component analysis (PCA) and hierarchical cluster analysis (HCA) of a random sample of 170 followers. User emphasis of keywords remained across levels but decreased by 9.5 percentage points. PCA and HCA identified 12 statistically unique clusters of followers within the @SafetyMD Twitter network. This study is one of the first to develop methods for use by stakeholders to evaluate and optimize their use of Twitter to disseminate health information. Our new methods provide preliminary evidence that individual stakeholders can evaluate the effectiveness of health information dissemination and create content-specific clusters for more specific targeted messaging.

  6. Myeloperoxidase in the plasma and placenta of normal pregnant women and women with pregnancies complicated by preeclampsia and intrauterine growth restriction.

    PubMed

    Hung, T-H; Chen, S-F; Lo, L-M; Li, M-J; Yeh, Y-L; Hsieh, T-T

    2012-04-01

    Myeloperoxidase (MPO) is a heme protein produced and released by activated neutrophils and monocytes, and increased MPO is considered important in the pathophysiology of cardiovascular diseases (CVD). Accumulating evidence suggests that preeclampsia (PE), idiopathic intrauterine growth restriction (IUGR), and CVD share many similar metabolic disturbances, including an enhanced systemic inflammatory response and endothelial dysfunction. We hypothesized that MPO plays an important role in the development of PE and IUGR. Plasma samples were collected mid-gestation and at delivery from women with normal pregnancies (n = 40) and those who subsequently developed PE (n = 20), IUGR (n = 11) or both (PE + IUGR, n = 8). Placental samples were obtained immediately after delivery from 22 women with normal pregnancies, 19 women with PE, 14 women with IUGR, and 14 women with PE + IUGR. The MPO concentrations were measured using ELISA. Women with PE + IUGR had significantly higher plasma MPO before delivery than normal pregnant women. There was no difference in plasma levels at mid-gestation or the placental concentrations between women with normal pregnancies and those who developed PE, IUGR, or PE + IUGR. Using explants prepared from the placentas of 8 women with normal pregnancies and 8 women with PE, we found no difference in the levels of MPO in the tissue homogenates and culture media between these two groups of women. Together, these results indicate that increased maternal circulating MPO in women with PE + IUGR is likely a result of enhanced systemic inflammation caused by the established disease rather than a primary pathophysiological factor. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

    PubMed Central

    Butts, Arielle; DeJarnette, Christian; Peters, Tracy L.; Parker, Josie E.; Kerns, Morgan E.; Eberle, Karen E.; Kelly, Steve L.

    2017-01-01

    ABSTRACT Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second

  8. Targeted polypeptide degradation

    DOEpatents

    Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

    2008-05-13

    This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

  9. Motion prediction of a non-cooperative space target

    NASA Astrophysics Data System (ADS)

    Zhou, Bang-Zhao; Cai, Guo-Ping; Liu, Yun-Meng; Liu, Pan

    2018-01-01

    Capturing a non-cooperative space target is a tremendously challenging research topic. Effective acquisition of motion information of the space target is the premise to realize target capture. In this paper, motion prediction of a free-floating non-cooperative target in space is studied and a motion prediction algorithm is proposed. In order to predict the motion of the free-floating non-cooperative target, dynamic parameters of the target must be firstly identified (estimated), such as inertia, angular momentum and kinetic energy and so on; then the predicted motion of the target can be acquired by substituting these identified parameters into the Euler's equations of the target. Accurate prediction needs precise identification. This paper presents an effective method to identify these dynamic parameters of a free-floating non-cooperative target. This method is based on two steps, (1) the rough estimation of the parameters is computed using the motion observation data to the target, and (2) the best estimation of the parameters is found by an optimization method. In the optimization problem, the objective function is based on the difference between the observed and the predicted motion, and the interior-point method (IPM) is chosen as the optimization algorithm, which starts at the rough estimate obtained in the first step and finds a global minimum to the objective function with the guidance of objective function's gradient. So the speed of IPM searching for the global minimum is fast, and an accurate identification can be obtained in time. The numerical results show that the proposed motion prediction algorithm is able to predict the motion of the target.

  10. Controversies in targeted therapy of adult T cell leukemia/lymphoma: ON target or OFF target effects?

    PubMed

    Nasr, Rihab; El Hajj, Hiba; Kfoury, Youmna; de Thé, Hugues; Hermine, Olivier; Bazarbachi, Ali

    2011-06-01

    Adult T cell leukemia/lymphoma (ATL) represents an ideal model for targeted therapy because of intrinsic chemo-resistance of ATL cells and the presence of two well identified targets: the HTLV-I retrovirus and the viral oncoprotein Tax. The combination of zidovudine (AZT) and interferon-alpha (IFN) has a dramatic impact on survival of ATL patients. Although the mechanism of action remains unclear, arguments in favor or against a direct antiviral effect will be discussed. Yet, most patients relapse and alternative therapies are mandatory. IFN and arsenic trioxide induce Tax proteolysis, synergize to induce apoptosis in ATL cells and cure Tax-driven ATL in mice through specific targeting of leukemia initiating cell activity. These results provide a biological basis for the clinical success of arsenic/IFN/AZT therapy in ATL patients and suggest that both extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) are needed to cure ATL.

  11. Utilizing random Forest QSAR models with optimized parameters for target identification and its application to target-fishing server.

    PubMed

    Lee, Kyoungyeul; Lee, Minho; Kim, Dongsup

    2017-12-28

    The identification of target molecules is important for understanding the mechanism of "target deconvolution" in phenotypic screening and "polypharmacology" of drugs. Because conventional methods of identifying targets require time and cost, in-silico target identification has been considered an alternative solution. One of the well-known in-silico methods of identifying targets involves structure activity relationships (SARs). SARs have advantages such as low computational cost and high feasibility; however, the data dependency in the SAR approach causes imbalance of active data and ambiguity of inactive data throughout targets. We developed a ligand-based virtual screening model comprising 1121 target SAR models built using a random forest algorithm. The performance of each target model was tested by employing the ROC curve and the mean score using an internal five-fold cross validation. Moreover, recall rates for top-k targets were calculated to assess the performance of target ranking. A benchmark model using an optimized sampling method and parameters was examined via external validation set. The result shows recall rates of 67.6% and 73.9% for top-11 (1% of the total targets) and top-33, respectively. We provide a website for users to search the top-k targets for query ligands available publicly at http://rfqsar.kaist.ac.kr . The target models that we built can be used for both predicting the activity of ligands toward each target and ranking candidate targets for a query ligand using a unified scoring scheme. The scores are additionally fitted to the probability so that users can estimate how likely a ligand-target interaction is active. The user interface of our web site is user friendly and intuitive, offering useful information and cross references.

  12. Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

    PubMed

    Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang Ping; Kim, Songmi; Arulalapperumal, Venkatesh; Lee, Keun Woo

    2015-07-01

    Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors. © 2014 Wiley Periodicals, Inc.

  13. Identifying targets for improvement in nighttime seat belt use : traffic tech.

    DOT National Transportation Integrated Search

    2010-07-01

    Previous research has found that the proportion of unbelted : fatalities is greater at night than during the day. Failure to use : seat belts has been identified as an important factor in nighttime : fatalities, yet there are still many gaps in our k...

  14. Walking the interactome to identify human miRNA-disease associations through the functional link between miRNA targets and disease genes

    PubMed Central

    2013-01-01

    Background MicroRNAs (miRNAs) are important post-transcriptional regulators that have been demonstrated to play an important role in human diseases. Elucidating the associations between miRNAs and diseases at the systematic level will deepen our understanding of the molecular mechanisms of diseases. However, miRNA-disease associations identified by previous computational methods are far from completeness and more effort is needed. Results We developed a computational framework to identify miRNA-disease associations by performing random walk analysis, and focused on the functional link between miRNA targets and disease genes in protein-protein interaction (PPI) networks. Furthermore, a bipartite miRNA-disease network was constructed, from which several miRNA-disease co-regulated modules were identified by hierarchical clustering analysis. Our approach achieved satisfactory performance in identifying known cancer-related miRNAs for nine human cancers with an area under the ROC curve (AUC) ranging from 71.3% to 91.3%. By systematically analyzing the global properties of the miRNA-disease network, we found that only a small number of miRNAs regulated genes involved in various diseases, genes associated with neurological diseases were preferentially regulated by miRNAs and some immunological diseases were associated with several specific miRNAs. We also observed that most diseases in the same co-regulated module tended to belong to the same disease category, indicating that these diseases might share similar miRNA regulatory mechanisms. Conclusions In this study, we present a computational framework to identify miRNA-disease associations, and further construct a bipartite miRNA-disease network for systematically analyzing the global properties of miRNA regulation of disease genes. Our findings provide a broad perspective on the relationships between miRNAs and diseases and could potentially aid future research efforts concerning miRNA involvement in disease pathogenesis

  15. Identifying natural compounds as multi-target-directed ligands against Alzheimer's disease: an in silico approach.

    PubMed

    Ambure, Pravin; Bhat, Jyotsna; Puzyn, Tomasz; Roy, Kunal

    2018-04-23

    Alzheimer's disease (AD) is a multi-factorial disease, which can be simply outlined as an irreversible and progressive neurodegenerative disorder with an unclear root cause. It is a major cause of dementia in old aged people. In the present study, utilizing the structural and biological activity information of ligands for five important and mostly studied vital targets (i.e. cyclin-dependant kinase 5, β-secretase, monoamine oxidase B, glycogen synthase kinase 3β, acetylcholinesterase) that are believed to be effective against AD, we have developed five classification models using linear discriminant analysis (LDA) technique. Considering the importance of data curation, we have given more attention towards the chemical and biological data curation, which is a difficult task especially in case of big data-sets. Thus, to ease the curation process we have designed Konstanz Information Miner (KNIME) workflows, which are made available at http://teqip.jdvu.ac.in/QSAR_Tools/ . The developed models were appropriately validated based on the predictions for experiment derived data from test sets, as well as true external set compounds including known multi-target compounds. The domain of applicability for each classification model was checked based on a confidence estimation approach. Further, these validated models were employed for screening of natural compounds collected from the InterBioScreen natural database ( https://www.ibscreen.com/natural-compounds ). Further, the natural compounds that were categorized as 'actives' in at least two classification models out of five developed models were considered as multi-target leads, and these compounds were further screened using the drug-like filter, molecular docking technique and then thoroughly analyzed using molecular dynamics studies. Finally, the most potential multi-target natural compounds against AD are suggested.

  16. Genomic Target Database (GTD): A database of potential targets in human pathogenic bacteria

    PubMed Central

    Barh, Debmalya; Kumar, Anil; Misra, Amarendra Narayana

    2009-01-01

    A Genomic Target Database (GTD) has been developed having putative genomic drug targets for human bacterial pathogens. The selected pathogens are either drug resistant or vaccines are yet to be developed against them. The drug targets have been identified using subtractive genomics approaches and these are subsequently classified into Drug targets in pathogen specific unique metabolic pathways,Drug targets in host-pathogen common metabolic pathways, andMembrane localized drug targets. HTML code is used to link each target to its various properties and other available public resources. Essential resources and tools for subtractive genomic analysis, sub-cellular localization, vaccine and drug designing are also mentioned. To the best of authors knowledge, no such database (DB) is presently available that has listed metabolic pathways and membrane specific genomic drug targets based on subtractive genomics. Listed targets in GTD are readily available resource in developing drug and vaccine against the respective pathogen, its subtypes, and other family members. Currently GTD contains 58 drug targets for four pathogens. Shortly, drug targets for six more pathogens will be listed. Availability GTD is available at IIOAB website http://www.iioab.webs.com/GTD.htm. It can also be accessed at http://www.iioabdgd.webs.com.GTD is free for academic research and non-commercial use only. Commercial use is strictly prohibited without prior permission from IIOAB. PMID:20011153

  17. Where to Go Next? Identifying Target Areas in the North Atlantic for Future Seafloor Mapping Initiatives

    NASA Astrophysics Data System (ADS)

    Woelfl, A. C.; Jencks, J.; Johnston, G.; Varner, J. D.; Devey, C. W.

    2017-12-01

    Human activities are rapidly expanding into the oceans, yet detailed bathymetric maps do not exist for most of the seafloor that would permit governments to formulate sensible usage rules. Changing this situation will require an enormous international mapping effort. To ensure that this effort is directed towards the regions most in need of mapping, we need to know which areas have already been mapped and which areas are potentially most interesting. Despite various mapping efforts in recent years, large parts of the Atlantic still lack detailed bathymetric information. To successfully plan for future mapping efforts to fill these gaps, knowledge of current data coverage is imperative to avoid duplication of effort. While certain datasets are publically available online (e.g. NOAA's NCEI, EMODnet, IHO-DCDB, LDEO's GMRT), many are not. However, with the limited information we do have at hand, the question remains, where should we map next? And what criteria should we take into account? In 2016, a study was taken on as part of the efforts of the International Atlantic Seabed Mapping Working Group (ASMIWG). The ASMIWG, established by the Tri-Partite Galway Statement Implementation Committee, was tasked to develop a cohesive seabed mapping strategy for the Atlantic Ocean. The aim of our study was to develop a reproducible process for identifying and evaluating potential target areas within the North Atlantic that represent suitable sites for future bathymetric surveys. The sites were selected by applying a GIS-based suitability analysis that included specific user group-based parameters of the marine environment. Furthermore, information regarding current data coverage were gathered to take into account in the selection process. The results reveal the suitability of sites within the North Atlantic based on the selected criteria. Three potential target sites should be seen as flexible suggestions for future mapping initiatives rather than a rigid, defined set of areas

  18. Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis

    PubMed Central

    Parker, Robert; Siddiqui, Nazneen; Potiphar, Lee; Goldin, Rob; Timms, John F.; Wells, Athol U.; Kon, Onn M.; Wickremasinghe, Melissa; Mitchell, Donald; Weeks, Mark E.; Lalvani, Ajit

    2017-01-01

    Background Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. Methods Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. Results We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin

  19. Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

    DOE PAGES

    Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

    2017-07-19

    Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

  20. Using Targeting Outcomes of Programs as a Framework to Target Photographic Events in Nonformal Educational Programs

    ERIC Educational Resources Information Center

    Rockwell, S. Kay; Albrecht, Julie A.; Nugent, Gwen C.; Kunz, Gina M.

    2012-01-01

    Targeting Outcomes of Programs (TOP) is a seven-step hierarchical programming model in which the program development and performance sides are mirror images of each other. It served as a framework to identify a simple method for targeting photographic events in nonformal education programs, indicating why, when, and how photographs would be useful…

  1. New target for inhibition of bacterial RNA polymerase: 'switch region'.

    PubMed

    Srivastava, Aashish; Talaue, Meliza; Liu, Shuang; Degen, David; Ebright, Richard Y; Sineva, Elena; Chakraborty, Anirban; Druzhinin, Sergey Y; Chatterjee, Sujoy; Mukhopadhyay, Jayanta; Ebright, Yon W; Zozula, Alex; Shen, Juan; Sengupta, Sonali; Niedfeldt, Rui Rong; Xin, Cai; Kaneko, Takushi; Irschik, Herbert; Jansen, Rolf; Donadio, Stefano; Connell, Nancy; Ebright, Richard H

    2011-10-01

    A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Identifying microRNA/mRNA dysregulations in ovarian cancer

    PubMed Central

    2012-01-01

    Background MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). Methods TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. Results We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory

  3. Identifying microRNA/mRNA dysregulations in ovarian cancer.

    PubMed

    Miles, Gregory D; Seiler, Michael; Rodriguez, Lorna; Rajagopal, Gunaretnam; Bhanot, Gyan

    2012-03-27

    MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Our findings identify

  4. Uveal Melanoma: Identifying Immunological and Chemotherapeutic Targets to Treat Metastases.

    PubMed

    Jager, Martine J; Dogrusöz, Mehmet; Woodman, Scott E

    2017-01-01

    Uveal melanoma is an intraocular malignancy that, depending on its size and genetic make-up, may lead to metastases in up to 50% of cases. Currently, no therapy has been proven to improve survival. However, new therapies exploiting immune responses against metastases are being developed. The primary tumor is well characterized: tumors at high risk of developing metastases often contain macrophages and lymphocytes. However, these lymphocytes are often regulatory T cells that may suppress immune response. Currently, immune checkpoint inhibitors have shown marked efficacy in multiple cancers (eg, cutaneous melanoma) but do not yet improve survival in uveal melanoma patients. More knowledge needs to be acquired regarding the function of T cells in uveal melanoma. Other therapeutic options are related to the biochemical pathways. Targeting the RAF-MEK-ERK pathway with small molecule MEK inhibitors abrogates the growth of UM cells harboring GNAQ/GNA11 Q209 mutations, suggesting that these aberrant G-protein oncogenes mediate, at least in part, their effect through this hallmark proliferation pathway. Other pathways are also implicated, such as those involving c-Jun and YAP. Further studies may show how interference in the different pathways may affect survival. Copyright 2017 Asia-Pacific Academy of Ophthalmology.

  5. Systems biology combining human- and animal-data miRNA and mRNA data identifies new targets in ureteropelvic junction obstruction.

    PubMed

    Papadopoulos, Theofilos; Casemayou, Audrey; Neau, Eric; Breuil, Benjamin; Caubet, Cécile; Calise, Denis; Thornhill, Barbara A; Bachvarova, Magdalena; Belliere, Julie; Chevalier, Robert L; Moulos, Panagiotis; Bachvarov, Dimcho; Buffin-Meyer, Benedicte; Decramer, Stéphane; Auriol, Françoise Conte; Bascands, Jean-Loup; Schanstra, Joost P; Klein, Julie

    2017-03-01

    Although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. We have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.

  6. Controversies in Targeted Therapy of Adult T Cell Leukemia/Lymphoma: ON Target or OFF Target Effects?

    PubMed Central

    Nasr, Rihab; Hajj, Hiba El; Kfoury, Youmna; de Thé, Hugues; Hermine, Olivier; Bazarbachi, Ali

    2011-01-01

    Adult T cell leukemia/lymphoma (ATL) represents an ideal model for targeted therapy because of intrinsic chemo-resistance of ATL cells and the presence of two well identified targets: the HTLV-I retrovirus and the viral oncoprotein Tax. The combination of zidovudine (AZT) and interferon-alpha (IFN) has a dramatic impact on survival of ATL patients. Although the mechanism of action remains unclear, arguments in favor or against a direct antiviral effect will be discussed. Yet, most patients relapse and alternative therapies are mandatory. IFN and arsenic trioxide induce Tax proteolysis, synergize to induce apoptosis in ATL cells and cure Tax-driven ATL in mice through specific targeting of leukemia initiating cell activity. These results provide a biological basis for the clinical success of arsenic/IFN/AZT therapy in ATL patients and suggest that both extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) are needed to cure ATL. PMID:21994752

  7. Characterizing and Targeting Replication Stress Response Defects in Breast Cancer

    DTIC Science & Technology

    2013-08-01

    This project is to use cutting-edge technologies to characterize novel RSR genes and their functions in tumor suppression; identify gene signature...and membrane proteins associated with defective RSR; identify drugs that target these defects; and develop RSR-defect-targeting nanoparticles for...screening and validation of drugs that target RSR-defect cells. The progress of our third year research is described below. BODY The tasks

  8. A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

    PubMed

    Garcia-Martinez, Irma; Weiss, Theresa R; Yousaf, Muhammad N; Ali, Ather; Mehal, Wajahat Z

    2018-01-01

    Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

  9. A Target-Based Whole Cell Screen Approach To Identify Potential Inhibitors of Mycobacterium tuberculosis Signal Peptidase

    PubMed Central

    2016-01-01

    The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane. During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We conducted a limited structure–activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 μM against the LepB-UE strain and 18 μM against the wt); several analogues were less potent against the LepB overexpressing strain. A number of chemical modifications around the hydrazone moiety resulted in improved potency. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism. PMID:27642770

  10. Identification of tissue-specific targeting peptide

    NASA Astrophysics Data System (ADS)

    Jung, Eunkyoung; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Seung-Hoon; Kim, Daejin; Park, Kisoo; Choi, Kihang; Choi, Yun-Jaie; Jung, Dong Hyun

    2012-11-01

    Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

  11. Targeted Deep Resequencing Identifies Coding Variants in the PEAR1 Gene That Play a Role in Platelet Aggregation

    PubMed Central

    Kim, Yoonhee; Suktitipat, Bhoom; Yanek, Lisa R.; Faraday, Nauder; Wilson, Alexander F.; Becker, Diane M.; Becker, Lewis C.; Mathias, Rasika A.

    2013-01-01

    Platelet aggregation is heritable, and genome-wide association studies have detected strong associations with a common intronic variant of the platelet endothelial aggregation receptor1 (PEAR1) gene both in African American and European American individuals. In this study, we used a sequencing approach to identify additional exonic variants in PEAR1 that may also determine variability in platelet aggregation in the GeneSTAR Study. A 0.3 Mb targeted region on chromosome 1q23.1 including the entire PEAR1 gene was Sanger sequenced in 104 subjects (45% male, 49% African American, age = 52±13) selected on the basis of hyper- and hypo- aggregation across three different agonists (collagen, epinephrine, and adenosine diphosphate). Single-variant and multi-variant burden tests for association were performed. Of the 235 variants identified through sequencing, 61 were novel, and three of these were missense variants. More rare variants (MAF<5%) were noted in African Americans compared to European Americans (108 vs. 45). The common intronic GWAS-identified variant (rs12041331) demonstrated the most significant association signal in African Americans (p = 4.020×10−4); no association was seen for additional exonic variants in this group. In contrast, multi-variant burden tests indicated that exonic variants play a more significant role in European Americans (p = 0.0099 for the collective coding variants compared to p = 0.0565 for intronic variant rs12041331). Imputation of the individual exonic variants in the rest of the GeneSTAR European American cohort (N = 1,965) supports the results noted in the sequenced discovery sample: p = 3.56×10−4, 2.27×10−7, 5.20×10−5 for coding synonymous variant rs56260937 and collagen, epinephrine and adenosine diphosphate induced platelet aggregation, respectively. Sequencing approaches confirm that a common intronic variant has the strongest association with platelet aggregation in African Americans, and

  12. Using experimental evolution to identify druggable targets that could inhibit the evolution of antimicrobial resistance

    PubMed Central

    Mehta, Heer H.; Prater, Amy G.; Shamoo, Yousif

    2017-01-01

    With multi-drug and pan-drug resistant bacteria becoming increasingly common in hospitals, antibiotic resistance has threatened to return us to a pre-antibiotic era that would completely undermine modern medicine. There is an urgent need to develop new antibiotics and strategies to combat resistance that are substantially different from earlier drug discovery efforts. One such strategy that would complement current and future antibiotics would be a class of co-drugs that target the evolution of resistance and thereby extend the efficacy of specific classes of antibiotics. A critical step in the development of such strategies lies in understanding the critical evolutionary trajectories responsible for resistance and which proteins or biochemical pathways within those trajectories would be good candidates for co-drug discovery. We identify the most important steps in the evolution of resistance for a specific pathogen and antibiotic combination by evolving highly polymorphic populations of pathogens to resistance in a novel bioreactor that favors biofilm development. As the populations evolve to increasing drug concentrations, we use deep sequencing to elucidate the network of genetic changes responsible for resistance and subsequent in vitro biochemistry and often structure determination to determine how the adaptive mutations produce resistance. Importantly, the identification of the molecular steps, their frequency within the populations and their chronology within the evolutionary trajectory toward resistance is critical to assessing their relative importance. In this work, we discuss findings from the evolution of the ESKAPE pathogen, Pseudomonas aeruginosa to the drug of last resort, colistin to illustrate the power of this approach. PMID:28928474

  13. Identifying HIV most-at-risk groups in Malawi for targeted interventions. A classification tree model.

    PubMed

    Emina, Jacques B O; Madise, Nyovani; Kuepie, Mathias; Zulu, Eliya M; Ye, Yazoume

    2013-05-28

    To identify HIV-socioeconomic predictors as well as the most-at-risk groups of women in Malawi. A cross-sectional survey. Malawi The study used a sample of 6395 women aged 15-49 years from the 2010 Malawi Health and Demographic Surveys. Individual HIV status: positive or not. Findings from the Pearson χ(2) and χ(2) Automatic Interaction Detector analyses revealed that marital status is the most significant predictor of HIV. Women who are no longer in union and living in the highest wealth quintiles households constitute the most-at-risk group, whereas the less-at-risk group includes young women (15-24) never married or in union and living in rural areas. In the light of these findings, this study recommends: (1) that the design and implementation of targeted interventions should consider the magnitude of HIV prevalence and demographic size of most-at-risk groups. Preventive interventions should prioritise couples and never married people aged 25-49 years and living in rural areas because this group accounts for 49% of the study population and 40% of women living with HIV in Malawi; (2) with reference to treatment and care, higher priority must be given to promoting HIV test, monitoring and evaluation of equity in access to treatment among women in union disruption and never married or women in union aged 30-49 years and living in urban areas; (3) community health workers, households-based campaign, reproductive-health services and reproductive-health courses at school could be used as canons to achieve universal prevention strategy, testing, counselling and treatment.

  14. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    PubMed

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  15. HomoTarget: a new algorithm for prediction of microRNA targets in Homo sapiens.

    PubMed

    Ahmadi, Hamed; Ahmadi, Ali; Azimzadeh-Jamalkandi, Sadegh; Shoorehdeli, Mahdi Aliyari; Salehzadeh-Yazdi, Ali; Bidkhori, Gholamreza; Masoudi-Nejad, Ali

    2013-02-01

    MiRNAs play an essential role in the networks of gene regulation by inhibiting the translation of target mRNAs. Several computational approaches have been proposed for the prediction of miRNA target-genes. Reports reveal a large fraction of under-predicted or falsely predicted target genes. Thus, there is an imperative need to develop a computational method by which the target mRNAs of existing miRNAs can be correctly identified. In this study, combined pattern recognition neural network (PRNN) and principle component analysis (PCA) architecture has been proposed in order to model the complicated relationship between miRNAs and their target mRNAs in humans. The results of several types of intelligent classifiers and our proposed model were compared, showing that our algorithm outperformed them with higher sensitivity and specificity. Using the recent release of the mirBase database to find potential targets of miRNAs, this model incorporated twelve structural, thermodynamic and positional features of miRNA:mRNA binding sites to select target candidates. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Integrative Analysis of Genetic, Genomic, and Phenotypic Data for Ethanol Behaviors: A Network-Based Pipeline for Identifying Mechanisms and Potential Drug Targets.

    PubMed

    Bogenpohl, James W; Mignogna, Kristin M; Smith, Maren L; Miles, Michael F

    2017-01-01

    Complex behavioral traits, such as alcohol abuse, are caused by an interplay of genetic and environmental factors, producing deleterious functional adaptations in the central nervous system. The long-term behavioral consequences of such changes are of substantial cost to both the individual and society. Substantial progress has been made in the last two decades in understanding elements of brain mechanisms underlying responses to ethanol in animal models and risk factors for alcohol use disorder (AUD) in humans. However, treatments for AUD remain largely ineffective and few medications for this disease state have been licensed. Genome-wide genetic polymorphism analysis (GWAS) in humans, behavioral genetic studies in animal models and brain gene expression studies produced by microarrays or RNA-seq have the potential to produce nonbiased and novel insight into the underlying neurobiology of AUD. However, the complexity of such information, both statistical and informational, has slowed progress toward identifying new targets for intervention in AUD. This chapter describes one approach for integrating behavioral, genetic, and genomic information across animal model and human studies. The goal of this approach is to identify networks of genes functioning in the brain that are most relevant to the underlying mechanisms of a complex disease such as AUD. We illustrate an example of how genomic studies in animal models can be used to produce robust gene networks that have functional implications, and to integrate such animal model genomic data with human genetic studies such as GWAS for AUD. We describe several useful analysis tools for such studies: ComBAT, WGCNA, and EW_dmGWAS. The end result of this analysis is a ranking of gene networks and identification of their cognate hub genes, which might provide eventual targets for future therapeutic development. Furthermore, this combined approach may also improve our understanding of basic mechanisms underlying gene x

  17. Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development.

    PubMed

    Ma, Jingqun; Brennan, Kaelan J; D'Aloia, Mitch R; Pascuzzi, Pete E; Weake, Vikki M

    2016-08-09

    The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia. Copyright © 2016 Ma et al.

  18. Application of Smoothing Techniques for Tracking Maneuvering Targets. Multiple Target Tracking in Clutter: New Approaches

    DTIC Science & Technology

    1992-07-01

    target state estimation is affected not only by the measurement noise but also by the uncertainty in the origins of the measurements. To improve the...to identify targets in the presence of anticipated background noise (including earth, lunar, star backgrounds, complicated spacecraft structures...each other. Futhermore, those frames are often degraded versions of the original scene due to blur and noise . Through the task of image registration

  19. T Cell Post-Transcriptional miRNA-mRNA Interaction Networks Identify Targets Associated with Susceptibility/Resistance to Collagen-induced Arthritis

    PubMed Central

    Macedo, Claudia; Cunha, Thiago M.; Nascimento, Daniele C. B.; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Cunha, Fernando Q.; Passos, Geraldo A.

    2013-01-01

    Background Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. Methodology/Principal Findings To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3+ T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir++ algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3′UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. Conclusions/Significance This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA. PMID:23359619

  20. T cell post-transcriptional miRNA-mRNA interaction networks identify targets associated with susceptibility/resistance to collagen-induced arthritis.

    PubMed

    Donate, Paula B; Fornari, Thais A; Macedo, Claudia; Cunha, Thiago M; Nascimento, Daniele C B; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Cunha, Fernando Q; Passos, Geraldo A

    2013-01-01

    Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+) T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++) algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.

  1. Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

    PubMed

    Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

    2016-08-01

    N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more

  2. A gain-of-function screen for genes that influence axon guidance identifies the NF-kappaB protein dorsal and reveals a requirement for the kinase Pelle in Drosophila photoreceptor axon targeting.

    PubMed

    Mindorff, Elizabeth N; O'Keefe, David D; Labbé, Alain; Yang, Jennie Ping; Ou, Yimiao; Yoshikawa, Shingo; van Meyel, Donald J

    2007-08-01

    To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. We mobilized the Gene Search (GS) P element and identified 42 lines with insertions in unique loci, including leak/roundabout2, which encodes an axon guidance receptor and confirms the utility of our screen. The genes we identified encode proteins of diverse classes, some acting near the cell surface and others in the cytoplasm or nucleus. We found that one GS line drove misexpression of the NF-kappaB transcription factor Dorsal, causing motor axons to bypass their correct termination sites. In the developing visual system, Dorsal misexpression also caused photoreceptor axons to reach incorrect positions within the optic lobe. This mistargeting occurred without observable changes of cell fate and correlated with localization of ectopic Dorsal in distal axons. We found that Dorsal and its inhibitor Cactus are expressed in photoreceptors, though neither was required for axon targeting. However, mutation analyses of genes known to act upstream of Dorsal revealed a requirement for the interleukin receptor-associated kinase family kinase Pelle for layer-specific targeting of photoreceptor axons, validating our screen as a means to identify new molecular determinants of nervous system development in vivo.

  3. Cooperative tumour cell membrane targeted phototherapy

    NASA Astrophysics Data System (ADS)

    Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

    2017-06-01

    The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

  4. Ultra-low field nuclear magnetic resonance and magnetic resonance imaging to discriminate and identify materials

    DOEpatents

    Kraus, Robert H.; Matlashov, Andrei N.; Espy, Michelle A.; Volegov, Petr L.

    2010-03-30

    An ultra-low magnetic field NMR system can non-invasively examine containers. Database matching techniques can then identify hazardous materials within the containers. Ultra-low field NMR systems are ideal for this purpose because they do not require large powerful magnets and because they can examine materials enclosed in conductive shells such as lead shells. The NMR examination technique can be combined with ultra-low field NMR imaging, where an NMR image is obtained and analyzed to identify target volumes. Spatial sensitivity encoding can also be used to identify target volumes. After the target volumes are identified the NMR measurement technique can be used to identify their contents.

  5. Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

    PubMed

    Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

    2017-10-01

    Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

  6. Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

    PubMed Central

    Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan

    2015-01-01

    T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799

  7. Target Recognition Using Neural Networks for Model Deformation Measurements

    NASA Technical Reports Server (NTRS)

    Ross, Richard W.; Hibler, David L.

    1999-01-01

    Optical measurements provide a non-invasive method for measuring deformation of wind tunnel models. Model deformation systems use targets mounted or painted on the surface of the model to identify known positions, and photogrammetric methods are used to calculate 3-D positions of the targets on the model from digital 2-D images. Under ideal conditions, the reflective targets are placed against a dark background and provide high-contrast images, aiding in target recognition. However, glints of light reflecting from the model surface, or reduced contrast caused by light source or model smoothness constraints, can compromise accurate target determination using current algorithmic methods. This paper describes a technique using a neural network and image processing technologies which increases the reliability of target recognition systems. Unlike algorithmic methods, the neural network can be trained to identify the characteristic patterns that distinguish targets from other objects of similar size and appearance and can adapt to changes in lighting and environmental conditions.

  8. A network analysis of the Chinese medicine Lianhua-Qingwen formula to identify its main effective components.

    PubMed

    Wang, Chun-Hua; Zhong, Yi; Zhang, Yan; Liu, Jin-Ping; Wang, Yue-Fei; Jia, Wei-Na; Wang, Guo-Cai; Li, Zheng; Zhu, Yan; Gao, Xiu-Mei

    2016-02-01

    Chinese medicine is known to treat complex diseases with multiple components and multiple targets. However, the main effective components and their related key targets and functions remain to be identified. Herein, a network analysis method was developed to identify the main effective components and key targets of a Chinese medicine, Lianhua-Qingwen Formula (LQF). The LQF is commonly used for the prevention and treatment of viral influenza in China. It is composed of 11 herbs, gypsum and menthol with 61 compounds being identified in our previous work. In this paper, these 61 candidate compounds were used to find their related targets and construct the predicted-target (PT) network. An influenza-related protein-protein interaction (PPI) network was constructed and integrated with the PT network. Then the compound-effective target (CET) network and compound-ineffective target network (CIT) were extracted, respectively. A novel approach was developed to identify effective components by comparing CET and CIT networks. As a result, 15 main effective components were identified along with 61 corresponding targets. 7 of these main effective components were further experimentally validated to have antivirus efficacy in vitro. The main effective component-target (MECT) network was further constructed with main effective components and their key targets. Gene Ontology (GO) analysis of the MECT network predicted key functions such as NO production being modulated by the LQF. Interestingly, five effective components were experimentally tested and exhibited inhibitory effects on NO production in the LPS induced RAW 264.7 cell. In summary, we have developed a novel approach to identify the main effective components in a Chinese medicine LQF and experimentally validated some of the predictions.

  9. Systemic approaches identify a garlic-derived chemical, Z-ajoene, as a glioblastoma multiforme cancer stem cell-specific targeting agent.

    PubMed

    Jung, Yuchae; Park, Heejoo; Zhao, Hui-Yuan; Jeon, Raok; Ryu, Jae-Ha; Kim, Woo-Young

    2014-07-01

    Glioblastoma multiforme (GBM) is one of the most common brain malignancies and has a very poor prognosis. Recent evidence suggests that the presence of cancer stem cells (CSC) in GBM and the rare CSC subpopulation that is resistant to chemotherapy may be responsible for the treatment failure and unfavorable prognosis of GBM. A garlic-derived compound, Z-ajoene, has shown a range of biological activities, including anti-proliferative effects on several cancers. Here, we demonstrated for the first time that Z-ajoene specifically inhibits the growth of the GBM CSC population. CSC sphere-forming inhibition was achieved at a concentration that did not exhibit a cytotoxic effect in regular cell culture conditions. The specificity of this inhibitory effect on the CSC population was confirmed by detecting CSC cell surface marker CD133 expression and biochemical marker ALDH activity. In addition, stem cell-related mRNA profiling and real-time PCR revealed the differential expression of CSC-specific genes, including Notch, Wnt, and Hedgehog, upon treatment with Z-ajoene. A proteomic approach, i.e., reverse-phase protein array (RPPA) and Western blot analysis, showed decreased SMAD4, p-AKT, 14.3.3 and FOXO3A expression. The protein interaction map (http://string-db.org/) of the identified molecules suggested that the AKT, ERK/p38 and TGFβ signaling pathways are key mediators of Z-ajoene's action, which affects the transcriptional network that includes FOXO3A. These biological and bioinformatic analyses collectively demonstrate that Z-ajoene is a potential candidate for the treatment of GBM by specifically targeting GBM CSCs. We also show how this systemic approach strengthens the identification of new therapeutic agents that target CSCs.

  10. Targeted in-gel MRM: a hypothesis driven approach for colorectal cancer biomarker discovery in human feces.

    PubMed

    Ang, Ching-Seng; Nice, Edouard C

    2010-09-03

    Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in both men and women. The fecal occult blood test is currently the first line method for CRC screening but has an unacceptably low sensitivity and specificity. Improved screening tests are therefore urgently required for early stage CRC screening. We have described a hypothesis-driven approach for a rapid biomarker discovery process whereby selected proteins previously implicated as colorectal cancer-associated proteins (CCAP), which can potentially be shed into the feces from a colorectal tumor, are targeted for excision from 1D-SDS-PAGE based on their predicted molecular weight followed by directed identification and relative quantification using multiple reaction monitoring (MRM). This approach can significantly reduce the time for clinical assay development with the added advantage that many proteins will have been validated by previous in vitro and/or in vivo studies. Sixty potential CCAPs were selected from the literature and appropriate MRM conditions were established for measurement of proteotypic peptides. Nineteen of these proteins were detected in the feces from a patient with colorectal cancer. Relative quantitation of these 19 CCAP across 5 CRC patients and 5 healthy volunteers were carried out, revealing hemoglobin, myeloperoxidase, S100A9, filamin A and l-plastin to be present only in the feces of CRC patients.

  11. Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells*

    PubMed Central

    Ko, Chia-Chen; Chen, Yun-Ju; Chen, Chih-Ta; Liu, Yu-Chih; Cheng, Fong-Chi; Hsu, Kai-Chao; Chow, Lu-Ping

    2014-01-01

    Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy. PMID:24962584

  12. Synergies and trade-offs in achieving global biodiversity targets.

    PubMed

    Di Marco, Moreno; Butchart, Stuart H M; Visconti, Piero; Buchanan, Graeme M; Ficetola, Gentile F; Rondinini, Carlo

    2016-02-01

    After their failure to achieve a significant reduction in the global rate of biodiversity loss by 2010, world governments adopted 20 new ambitious Aichi biodiversity targets to be met by 2020. Efforts to achieve one particular target can contribute to achieving others, but different targets may sometimes require conflicting solutions. Consequently, lack of strategic thinking might result, once again, in a failure to achieve global commitments to biodiversity conservation. We illustrate this dilemma by focusing on Aichi Target 11. This target requires an expansion of terrestrial protected area coverage, which could also contribute to reducing the loss of natural habitats (Target 5), reducing human-induced species decline and extinction (Target 12), and maintaining global carbon stocks (Target 15). We considered the potential impact of expanding protected areas to mitigate global deforestation and the consequences for the distribution of suitable habitat for >10,000 species of forest vertebrates (amphibians, birds, and mammals). We first identified places where deforestation might have the highest impact on remaining forests and then identified places where deforestation might have the highest impact on forest vertebrates (considering aggregate suitable habitat for species). Expanding protected areas toward locations with the highest deforestation rates (Target 5) or the highest potential loss of aggregate species' suitable habitat (Target 12) resulted in partially different protected area network configurations (overlapping with each other by about 73%). Moreover, the latter approach contributed to safeguarding about 30% more global carbon stocks than the former. Further investigation of synergies and trade-offs between targets would shed light on these and other complex interactions, such as the interaction between reducing overexploitation of natural resources (Targets 6, 7), controlling invasive alien species (Target 9), and preventing extinctions of native

  13. Photoaffinity labeling in target- and binding-site identification

    PubMed Central

    Smith, Ewan; Collins, Ian

    2015-01-01

    Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

  14. Identify Your Brand, Before You Market.

    ERIC Educational Resources Information Center

    Claggett, Laura

    2002-01-01

    Discusses marketing in special libraries and suggests that librarians need to identify library services that set them apart from others. Highlights include the competitive environment and alternatives for the consumer; value that the library offers; targeting consumers; return on investment; and determining why consumers choose your services. (LRW)

  15. PDK1/Akt/PDE4D axis identified as a target for asthma remedy synergistic with β2 AR agonists by a natural agent arctigenin.

    PubMed

    Fang, R; Cui, Q; Sun, J; Duan, X; Ma, X; Wang, W; Cheng, B; Liu, Y; Hou, Y; Bai, G

    2015-12-01

    Asthma is a heterogenetic disorder characterized by chronic inflammation with variable airflow obstruction and airway hyper-responsiveness. As the most potent and popular bronchodilators, β2 adrenergic receptor (β2 AR) agonists bind to the β2 ARs that are coupled via a stimulatory G protein to adenylyl cyclase, thereby improving cAMP accumulation and resulting in airway smooth muscle relaxation. We previously demonstrated arctigenin had a synergistic function with the β2 AR agonist, but the target for this remained elusive. Chemical proteomics capturing was used to enrich and uncover the target of arctigenin in human bronchial smooth muscle cells, and reverse docking and molecular dynamic stimulation were performed to evaluate the binding of arctigenin and its target. In vitro enzyme activities and protein levels were demonstrated with special kits and Western blotting. Finally, guinea pig tracheal muscle segregation and ex vivo function were analysed. Arctigenin bound to PDK1 with an ideal binding free energy -25.45 kcal/mol and inhibited PDK1 kinase activity without changing its protein level. Additionally, arctigenin reduced PKB/Akt-induced phosphorylation of PDE4D, which was first identified in this study. Attenuation of PDE4D resulted in cAMP accumulation in human bronchial smooth muscle. The inhibition of PDK1 showed a synergistic function with β2 AR agonists and relaxed the constriction of segregated guinea pig tracheal muscle. The PDK1/Akt/PDE4D axis serves as a novel asthma target, which may benefit airflow obstruction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

    PubMed

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Danielski, Lucineia G; Petronilho, Fabricia; Carvalho, André F; Quevedo, João

    2017-09-01

    Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol) or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against

  17. Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

    PubMed Central

    Tselepi, Maria; Gómez, Rodolfo; Woods, Steven; Hui, Wang; Smith, Graham R.; Shanley, Daryl P.; Clark, Ian M.; Young, David A.

    2015-01-01

    Abstract microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280 PMID:26175215

  18. Design of a Covert RFID Tag Network for Target Discovery and Target Information Routing

    PubMed Central

    Pan, Qihe; Narayanan, Ram M.

    2011-01-01

    Radio frequency identification (RFID) tags are small electronic devices working in the radio frequency range. They use wireless radio communications to automatically identify objects or people without the need for line-of-sight or contact, and are widely used in inventory tracking, object location, environmental monitoring. This paper presents a design of a covert RFID tag network for target discovery and target information routing. In the design, a static or very slowly moving target in the field of RFID tags transmits a distinct pseudo-noise signal, and the RFID tags in the network collect the target information and route it to the command center. A map of each RFID tag’s location is saved at command center, which can determine where a RFID tag is located based on each RFID tag’s ID. We propose the target information collection method with target association and clustering, and we also propose the information routing algorithm within the RFID tag network. The design and operation of the proposed algorithms are illustrated through examples. Simulation results demonstrate the effectiveness of the design. PMID:22163693

  19. Design of a covert RFID tag network for target discovery and target information routing.

    PubMed

    Pan, Qihe; Narayanan, Ram M

    2011-01-01

    Radio frequency identification (RFID) tags are small electronic devices working in the radio frequency range. They use wireless radio communications to automatically identify objects or people without the need for line-of-sight or contact, and are widely used in inventory tracking, object location, environmental monitoring. This paper presents a design of a covert RFID tag network for target discovery and target information routing. In the design, a static or very slowly moving target in the field of RFID tags transmits a distinct pseudo-noise signal, and the RFID tags in the network collect the target information and route it to the command center. A map of each RFID tag's location is saved at command center, which can determine where a RFID tag is located based on each RFID tag's ID. We propose the target information collection method with target association and clustering, and we also propose the information routing algorithm within the RFID tag network. The design and operation of the proposed algorithms are illustrated through examples. Simulation results demonstrate the effectiveness of the design.

  20. TargetMiner: microRNA target prediction with systematic identification of tissue-specific negative examples.

    PubMed

    Bandyopadhyay, Sanghamitra; Mitra, Ramkrishna

    2009-10-15

    Prediction of microRNA (miRNA) target mRNAs using machine learning approaches is an important area of research. However, most of the methods suffer from either high false positive or false negative rates. One reason for this is the marked deficiency of negative examples or miRNA non-target pairs. Systematic identification of non-target mRNAs is still not addressed properly, and therefore, current machine learning approaches are compelled to rely on artificially generated negative examples for training. In this article, we have identified approximately 300 tissue-specific negative examples using a novel approach that involves expression profiling of both miRNAs and mRNAs, miRNA-mRNA structural interactions and seed-site conservation. The newly generated negative examples are validated with pSILAC dataset, which elucidate the fact that the identified non-targets are indeed non-targets.These high-throughput tissue-specific negative examples and a set of experimentally verified positive examples are then used to build a system called TargetMiner, a support vector machine (SVM)-based classifier. In addition to assessing the prediction accuracy on cross-validation experiments, TargetMiner has been validated with a completely independent experimental test dataset. Our method outperforms 10 existing target prediction algorithms and provides a good balance between sensitivity and specificity that is not reflected in the existing methods. We achieve a significantly higher sensitivity and specificity of 69% and 67.8% based on a pool of 90 feature set and 76.5% and 66.1% using a set of 30 selected feature set on the completely independent test dataset. In order to establish the effectiveness of the systematically generated negative examples, the SVM is trained using a different set of negative data generated using the method in Yousef et al. A significantly higher false positive rate (70.6%) is observed when tested on the independent set, while all other factors are kept the

  1. Target Fishing for Chemical Compounds using Target-Ligand Activity data and Ranking based Methods

    PubMed Central

    Wale, Nikil; Karypis, George

    2009-01-01

    In recent years the development of computational techniques that identify all the likely targets for a given chemical compound, also termed as the problem of Target Fishing, has been an active area of research. Identification of likely targets of a chemical compound helps to understand problems such as toxicity, lack of efficacy in humans, and poor physical properties associated with that compound in the early stages of drug discovery. In this paper we present a set of techniques whose goal is to rank or prioritize targets in the context of a given chemical compound such that most targets that this compound may show activity against appear higher in the ranked list. These methods are based on our extensions to the SVM and Ranking Perceptron algorithms for this problem. Our extensive experimental study shows that the methods developed in this work outperform previous approaches by 2% to 60% under different evaluation criterions. PMID:19764745

  2. Targeted marketing and public health.

    PubMed

    Grier, Sonya A; Kumanyika, Shiriki

    2010-01-01

    Targeted marketing techniques, which identify consumers who share common needs or characteristics and position products or services to appeal to and reach these consumers, are now the core of all marketing and facilitate its effectiveness. However, targeted marketing, particularly of products with proven or potential adverse effects (e.g., tobacco, alcohol, entertainment violence, or unhealthful foods) to consumer segments defined as vulnerable raises complex concerns for public health. It is critical that practitioners, academics, and policy makers in marketing, public health, and other fields recognize and understand targeted marketing as a specific contextual influence on the health of children and adolescents and, for different reasons, ethnic minority populations and other populations who may benefit from public health protections. For beneficial products, such understanding can foster more socially productive targeting. For potentially harmful products, understanding the nature and scope of targeted marketing influences will support identification and implementation of corrective policies.

  3. Impacts of transgenic poplar-cotton agro-ecosystems upon target pests and non-target insects under field conditions.

    PubMed

    Zhang, D J; Liu, J X; Lu, Z Y; Li, C L; Comada, E; Yang, M S

    2015-07-27

    Poplar-cotton agro-ecosystems are the main agricultural planting modes of cotton fields in China. With increasing acres devoted to transgenic insect-resistant poplar and transgenic insect-resistant cotton, studies examining the effects of transgenic plants on target and non-target insects become increasingly important. We systematically surveyed populations of both target pests and non-target insects for 4 different combinations of poplar-cotton eco-systems over 3 years. Transgenic Bt cotton strongly resisted the target insects Fall webworm moth [Hyphantria cunea (Drury)], Sylepta derogata Fabrieius, and American bollworm (Heliothis armigera), but no clear impact on non-target insect cotton aphids (Aphis gossypii). Importantly, intercrops containing transgenic Pb29 poplar significantly increased the inhibitory effects of Bt cotton on Fall webworm moth in ecosystem IV. Highly resistant Pb29 poplar reduced populations of the target pests Grnsonoma minutara Hubner and non-target insect poplar leaf aphid (Chaitophorus po-pulialbae), while Fall webworm moth populations were unaffected. We determined the effects of Bt toxin from transgenic poplar and cotton on target and non-target pests in different ecosystems of cotton-poplar intercrops and identified the synergistic effects of such combinations toward both target and non-target insects.

  4. HPPD: ligand- and target-based virtual screening on a herbicide target.

    PubMed

    López-Ramos, Miriam; Perruccio, Francesca

    2010-05-24

    Hydroxyphenylpyruvate dioxygenase (HPPD) has proven to be a very successful target for the development of herbicides with bleaching properties, and today HPPD inhibitors are well established in the agrochemical market. Syngenta has a long history of HPPD-inhibitor research, and HPPD was chosen as a case study for the validation of diverse ligand- and target-based virtual screening approaches to identify compounds with inhibitory properties. Two-dimensional extended connectivity fingerprints, three-dimensional shape-based tools (ROCS, EON, and Phase-shape) and a pharmacophore approach (Phase) were used as ligand-based methods; Glide and Gold were used as target-based. Both the virtual screening utility and the scaffold-hopping ability of the screening tools were assessed. Particular emphasis was put on the specific pitfalls to take into account for the design of a virtual screening campaign in an agrochemical context, as compared to a pharmaceutical environment.

  5. Economic evaluation of targeted cancer interventions: critical review and recommendations.

    PubMed

    Elkin, Elena B; Marshall, Deborah A; Kulin, Nathalie A; Ferrusi, Ilia L; Hassett, Michael J; Ladabaum, Uri; Phillips, Kathryn A

    2011-10-01

    Scientific advances have improved our ability to target cancer interventions to individuals who will benefit most and spare the risks and costs to those who will derive little benefit or even be harmed. Several approaches are currently used for targeting interventions for cancer risk reduction, screening, and treatment, including risk prediction algorithms for identifying high-risk subgroups and diagnostic tests for tumor markers and germline genetic mutations. Economic evaluation can inform decisions about the use of targeted interventions, which may be more costly than traditional strategies. However, assessing the impact of a targeted intervention on costs and health outcomes requires explicit consideration of the method of targeting. In this study, we describe the importance of this principle by reviewing published cost-effectiveness analyses of targeted interventions in breast cancer. Few studies we identified explicitly evaluated the relationships among the method of targeting, the accuracy of the targeting test, and outcomes of the targeted intervention. Those that did found that characteristics of targeting tests had a substantial impact on outcomes. We posit that the method of targeting and the outcomes of a targeted intervention are inextricably linked and recommend that cost-effectiveness analyses of targeted interventions explicitly consider costs and outcomes of the method of targeting.

  6. Systemic Response to Microgravity: Utilizing GeneLab Datasets to Identify Molecular Targets for Future Hypotheses-Driven Spaceflight Studies

    NASA Technical Reports Server (NTRS)

    Beheshti, Afshin; Ray, Shayoni; Fogle, Homer; Berrios, Daniel C.; Costes, Sylvain V.

    2017-01-01

    subsequent impact on health, a theoretical health risk score was calculated. The genes and miRNAs identified from our analyses can be targeted for future research involving efficient countermeasure design. Our study thus exemplifies the utility of GeneLab data repository to aid in the process of performing novel hypothesis based spaceflight research aimed at elucidating the global impact of environmental stressors at multiple biological scales.

  7. Computational selection of antibody-drug conjugate targets for breast cancer

    PubMed Central

    Fauteux, François; Hill, Jennifer J.; Jaramillo, Maria L.; Pan, Youlian; Phan, Sieu; Famili, Fazel; O'Connor-McCourt, Maureen

    2016-01-01

    The selection of therapeutic targets is a critical aspect of antibody-drug conjugate research and development. In this study, we applied computational methods to select candidate targets overexpressed in three major breast cancer subtypes as compared with a range of vital organs and tissues. Microarray data corresponding to over 8,000 tissue samples were collected from the public domain. Breast cancer samples were classified into molecular subtypes using an iterative ensemble approach combining six classification algorithms and three feature selection techniques, including a novel kernel density-based method. This feature selection method was used in conjunction with differential expression and subcellular localization information to assemble a primary list of targets. A total of 50 cell membrane targets were identified, including one target for which an antibody-drug conjugate is in clinical use, and six targets for which antibody-drug conjugates are in clinical trials for the treatment of breast cancer and other solid tumors. In addition, 50 extracellular proteins were identified as potential targets for non-internalizing strategies and alternative modalities. Candidate targets linked with the epithelial-to-mesenchymal transition were identified by analyzing differential gene expression in epithelial and mesenchymal tumor-derived cell lines. Overall, these results show that mining human gene expression data has the power to select and prioritize breast cancer antibody-drug conjugate targets, and the potential to lead to new and more effective cancer therapeutics. PMID:26700623

  8. Method of identifying hairpin DNA probes by partial fold analysis

    DOEpatents

    Miller, Benjamin L [Penfield, NY; Strohsahl, Christopher M [Saugerties, NY

    2009-10-06

    Method of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

  9. Method of identifying hairpin DNA probes by partial fold analysis

    DOEpatents

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2008-10-28

    Methods of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

  10. Immunotherapy Targets Common Cancer Mutation

    Cancer.gov

    In a study of an immune therapy for colorectal cancer that involved a single patient, researchers identified a method for targeting the cancer-causing protein produced by a mutant form of the KRAS gene.

  11. Genomic approach to therapeutic target validation identifies a glucose-lowering GLP1R variant protective for coronary heart disease

    PubMed Central

    Scott, Robert A.; Freitag, Daniel F.; Li, Li; Chu, Audrey Y.; Surendran, Praveen; Young, Robin; Grarup, Niels; Stancáková, Alena; Chen, Yuning; V.Varga, Tibor; Yaghootkar, Hanieh; Luan, Jian'an; Zhao, Jing Hua; Willems, Sara M.; Wessel, Jennifer; Wang, Shuai; Maruthur, Nisa; Michailidou, Kyriaki; Pirie, Ailith; van der Lee, Sven J.; Gillson, Christopher; Olama, Ali Amin Al; Amouyel, Philippe; Arriola, Larraitz; Arveiler, Dominique; Aviles-Olmos, Iciar; Balkau, Beverley; Barricarte, Aurelio; Barroso, Inês; Garcia, Sara Benlloch; Bis, Joshua C.; Blankenberg, Stefan; Boehnke, Michael; Boeing, Heiner; Boerwinkle, Eric; Borecki, Ingrid B.; Bork-Jensen, Jette; Bowden, Sarah; Caldas, Carlos; Caslake, Muriel; Cupples, L. Adrienne; Cruchaga, Carlos; Czajkowski, Jacek; den Hoed, Marcel; Dunn, Janet A.; Earl, Helena M.; Ehret, Georg B.; Ferrannini, Ele; Ferrieres, Jean; Foltynie, Thomas; Ford, Ian; Forouhi, Nita G.; Gianfagna, Francesco; Gonzalez, Carlos; Grioni, Sara; Hiller, Louise; Jansson, Jan-Håkan; Jørgensen, Marit E.; Jukema, J. Wouter; Kaaks, Rudolf; Kee, Frank; Kerrison, Nicola D.; Key, Timothy J.; Kontto, Jukka; Kote-Jarai, Zsofia; Kraja, Aldi T.; Kuulasmaa, Kari; Kuusisto, Johanna; Linneberg, Allan; Liu, Chunyu; Marenne, Gaëlle; Mohlke, Karen L.; Morris, Andrew P.; Muir, Kenneth; Müller-Nurasyid, Martina; Munroe, Patricia B.; Navarro, Carmen; Nielsen, Sune F.; Nilsson, Peter M.; Nordestgaard, Børge G.; Packard, Chris J.; Palli, Domenico; Panico, Salvatore; Peloso, Gina M.; Perola, Markus; Peters, Annette; Poole, Christopher J.; Quirós, J. Ramón; Rolandsson, Olov; Sacerdote, Carlotta; Salomaa, Veikko; Sánchez, María-José; Sattar, Naveed; Sharp, Stephen J.; Sims, Rebecca; Slimani, Nadia; Smith, Jennifer A.; Thompson, Deborah J.; Trompet, Stella; Tumino, Rosario; van der A, Daphne L.; van der Schouw, Yvonne T.; Virtamo, Jarmo; Walker, Mark; Walter, Klaudia; Abraham, Jean E.; Amundadottir, Laufey T.; Aponte, Jennifer L.; Butterworth, Adam S.; Dupuis, Josée; Easton, Douglas F.; Eeles, Rosalind A.; Erdmann, Jeanette; Franks, Paul W.; Frayling, Timothy M.; Hansen, Torben; Howson, Joanna M. M.; Jørgensen, Torben; Kooner, Jaspal; Laakso, Markku; Langenberg, Claudia; McCarthy, Mark I.; Pankow, James S.; Pedersen, Oluf; Riboli, Elio; Rotter, Jerome I.; Saleheen, Danish; Samani, Nilesh J.; Schunkert, Heribert; Vollenweider, Peter; O'Rahilly, Stephen; Deloukas, Panos; Danesh, John; Goodarzi, Mark O.; Kathiresan, Sekar; Meigs, James B.; Ehm, Margaret G.; Wareham, Nicholas J.; Waterworth, Dawn M.

    2016-01-01

    Regulatory authorities have indicated that new drugs to treat type 2 diabetes (T2D) should not be associated with an unacceptable increase in cardiovascular risk. Human genetics may be able to inform development of antidiabetic therapies by predicting cardiovascular and other health endpoints. We therefore investigated the association of variants in 6 genes that encode drug targets for obesity or T2D with a range of metabolic traits in up to 11,806 individuals by targeted exome sequencing, and follow-up in 39,979 individuals by targeted genotyping, with additional in silico follow up in consortia. We used these data to first compare associations of variants in genes encoding drug targets with the effects of pharmacological manipulation of those targets in clinical trials. We then tested the association those variants with disease outcomes, including coronary heart disease, to predict cardiovascular safety of these agents. A low-frequency missense variant (Ala316Thr;rs10305492) in the gene encoding glucagon-like peptide-1 receptor (GLP1R), the target of GLP1R agonists, was associated with lower fasting glucose and lower T2D risk, consistent with GLP1R agonist therapies. The minor allele was also associated with protection against heart disease, thus providing evidence that GLP1R agonists are not likely to be associated with an unacceptable increase in cardiovascular risk. Our results provide an encouraging signal that these agents may be associated with benefit, a question currently being addressed in randomised controlled trials. Genetic variants associated with metabolic traits and multiple disease outcomes can be used to validate therapeutic targets at an early stage in the drug development process. PMID:27252175

  12. Identifying target processes for microbial electrosynthesis by elementary mode analysis.

    PubMed

    Kracke, Frauke; Krömer, Jens O

    2014-12-30

    Microbial electrosynthesis and electro fermentation are techniques that aim to optimize microbial production of chemicals and fuels by regulating the cellular redox balance via interaction with electrodes. While the concept is known for decades major knowledge gaps remain, which make it hard to evaluate its biotechnological potential. Here we present an in silico approach to identify beneficial production processes for electro fermentation by elementary mode analysis. Since the fundamentals of electron transport between electrodes and microbes have not been fully uncovered yet, we propose different options and discuss their impact on biomass and product yields. For the first time 20 different valuable products were screened for their potential to show increased yields during anaerobic electrically enhanced fermentation. Surprisingly we found that an increase in product formation by electrical enhancement is not necessarily dependent on the degree of reduction of the product but rather the metabolic pathway it is derived from. We present a variety of beneficial processes with product yield increases of maximal 36% in reductive and 84% in oxidative fermentations and final theoretical product yields up to 100%. This includes compounds that are already produced at industrial scale such as succinic acid, lysine and diaminopentane as well as potential novel bio-commodities such as isoprene, para-hydroxybenzoic acid and para-aminobenzoic acid. Furthermore, it is shown that the way of electron transport has major impact on achievable biomass and product yields. The coupling of electron transport to energy conservation could be identified as crucial for most processes. This study introduces a powerful tool to determine beneficial substrate and product combinations for electro-fermentation. It also highlights that the maximal yield achievable by bio electrochemical techniques depends strongly on the actual electron transport mechanisms. Therefore it is of great importance to

  13. Identifying patients in target customer segments using a two-stage clustering-classification approach: a hospital-based assessment.

    PubMed

    Chen, You-Shyang; Cheng, Ching-Hsue; Lai, Chien-Jung; Hsu, Cheng-Yi; Syu, Han-Jhou

    2012-02-01

    Identifying patients in a Target Customer Segment (TCS) is important to determine the demand for, and to appropriately allocate resources for, health care services. The purpose of this study is to propose a two-stage clustering-classification model through (1) initially integrating the RFM attribute and K-means algorithm for clustering the TCS patients and (2) then integrating the global discretization method and the rough set theory for classifying hospitalized departments and optimizing health care services. To assess the performance of the proposed model, a dataset was used from a representative hospital (termed Hospital-A) that was extracted from a database from an empirical study in Taiwan comprised of 183,947 samples that were characterized by 44 attributes during 2008. The proposed model was compared with three techniques, Decision Tree, Naive Bayes, and Multilayer Perceptron, and the empirical results showed significant promise of its accuracy. The generated knowledge-based rules provide useful information to maximize resource utilization and support the development of a strategy for decision-making in hospitals. From the findings, 75 patients in the TCS, three hospital departments, and specific diagnostic items were discovered in the data for Hospital-A. A potential determinant for gender differences was found, and the age attribute was not significant to the hospital departments. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets.

    PubMed

    McCutcheon, Krista M; Gray, Julia; Chen, Natalie Y; Liu, Keyi; Park, Minha; Ellsworth, Stote; Tripp, Ralph A; Tompkins, S Mark; Johnson, Scott K; Samet, Shelly; Pereira, Lenore; Kauvar, Lawrence M

    2014-01-01

    Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

  15. Tumor-targeting peptides from combinatorial libraries*

    PubMed Central

    Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S.

    2018-01-01

    Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges infighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. PMID:27210583

  16. Identifying HIV most-at-risk groups in Malawi for targeted interventions. A classification tree model

    PubMed Central

    Emina, Jacques B O; Madise, Nyovani; Kuepie, Mathias; Zulu, Eliya M; Ye, Yazoume

    2013-01-01

    Objectives To identify HIV-socioeconomic predictors as well as the most-at-risk groups of women in Malawi. Design A cross-sectional survey. Setting Malawi Participants The study used a sample of 6395 women aged 15–49 years from the 2010 Malawi Health and Demographic Surveys. Interventions N/A Primary and secondary outcome measures Individual HIV status: positive or not. Results Findings from the Pearson χ2 and χ2 Automatic Interaction Detector analyses revealed that marital status is the most significant predictor of HIV. Women who are no longer in union and living in the highest wealth quintiles households constitute the most-at-risk group, whereas the less-at-risk group includes young women (15–24) never married or in union and living in rural areas. Conclusions In the light of these findings, this study recommends: (1) that the design and implementation of targeted interventions should consider the magnitude of HIV prevalence and demographic size of most-at-risk groups. Preventive interventions should prioritise couples and never married people aged 25–49 years and living in rural areas because this group accounts for 49% of the study population and 40% of women living with HIV in Malawi; (2) with reference to treatment and care, higher priority must be given to promoting HIV test, monitoring and evaluation of equity in access to treatment among women in union disruption and never married or women in union aged 30–49 years and living in urban areas; (3) community health workers, households-based campaign, reproductive-health services and reproductive-health courses at school could be used as canons to achieve universal prevention strategy, testing, counselling and treatment. PMID:23793677

  17. [Thalidomide teratogenicity and its direct target identification].

    PubMed

    Ito, Takumi; Ando, Hideki; Handa, Hiroshi

    2015-01-01

    Half a century ago, thalidomide was developed as a sedative drug and was wildly used over 40 countries. However the drug has serious birth defects such as amelia and phocomelia. Now thalidomide is regarded as a clinically effective drug and used for the treatment of multiple myeloma under strict controls. The direct target of thalidomide had been a long-standing question. We identified cereblon as a primary direct target protein for thalidomide teratogenicity using new affinity bead technology in 2010. In this review, we introduce an overview of thalidomide teratogenicity, a story about how we identified cereblon, and recent advances in cereblon studies.

  18. INTEGRATIVE ANALYSIS OF GENETIC, GENOMIC AND PHENOTYPIC DATA FOR ETHANOL BEHAVIORS: A NETWORK-BASED PIPELINE FOR IDENTIFYING MECHANISMS AND POTENTIAL DRUG TARGETS

    PubMed Central

    Bogenpohl, James W.; Mignogna, Kristin M.; Smith, Maren L.; Miles, Michael F.

    2016-01-01

    Complex behavioral traits, such as alcohol abuse, are caused by an interplay of genetic and environmental factors, producing deleterious functional adaptations in the central nervous system. The long-term behavioral consequences of such changes are of substantial cost to both the individual and society. Substantial progress has been made in the last two decades in understanding elements of brain mechanisms underlying responses to ethanol in animal models and risk factors for alcohol use disorder (AUD) in humans. However, treatments for AUD remain largely ineffective and few medications for this disease state have been licensed. Genome-wide genetic polymorphism analysis (GWAS) in humans, behavioral genetic studies in animal models and brain gene expression studies produced by microarrays or RNA-seq have the potential to produce non-biased and novel insight into the underlying neurobiology of AUD. However, the complexity of such information, both statistical and informational, has slowed progress toward identifying new targets for intervention in AUD. This chapter describes one approach for integrating behavioral, genetic, and genomic information across animal model and human studies. The goal of this approach is to identify networks of genes functioning in the brain that are most relevant to the underlying mechanisms of a complex disease such as AUD. We illustrate an example of how genomic studies in animal models can be used to produce robust gene networks that have functional implications, and to integrate such animal model genomic data with human genetic studies such as GWAS for AUD. We describe several useful analysis tools for such studies: ComBAT, WGCNA and EW_dmGWAS. The end result of this analysis is a ranking of gene networks and identification of their cognate hub genes, which might provide eventual targets for future therapeutic development. Furthermore, this combined approach may also improve our understanding of basic mechanisms underlying gene x

  19. Loss of GPER identifies new targets for therapy among a subgroup of ERα-positive endometrial cancer patients with poor outcome

    PubMed Central

    Krakstad, C; Trovik, J; Wik, E; Engelsen, I B; Werner, H M J; Birkeland, E; Raeder, M B; Øyan, A M; Stefansson, I M; Kalland, K H; Akslen, L A; Salvesen, H B

    2012-01-01

    Background: The G protein-coupled oestrogen receptor, GPER, has been suggested as an alternative oestrogen receptor. Our purpose was to investigate the potential of GPER as a prognostic and predictive marker in endometrial carcinoma and to search for new drug candidates to improve treatment of aggressive disease. Materials and method: A total of 767 primary endometrial carcinomas derived from three patient series, including an external dataset, were studied for protein and mRNA expression levels to investigate and validate if GPER loss identifies poor prognosis and new targets for therapy in endometrial carcinoma. Gene expression levels, according to ERα/GPER status, were used to search the connectivity map database for small molecular inhibitors with potential for treatment of metastatic disease for receptor status subgroups. Results: Loss of GPER protein is significantly correlated with low GPER mRNA, high FIGO stage, non-endometrioid histology, high grade, aneuploidy and ERα loss (all P-values ⩽0.05). Loss of GPER among ERα-positive patients identifies a subgroup with poor prognosis that until now has been unrecognised, with reduced 5-year survival from 93% to 76% (P=0.003). Additional loss of GPER from primary to metastatic lesion counterparts further supports that loss of GPER is associated with disease progression. Conclusion: These results support that GPER status adds clinically relevant information to ERα status in endometrial carcinoma and suggest a potential for new inhibitors in the treatment of metastatic endometrial cancers with ERα expression and GPER loss. PMID:22415229

  20. Whole-Genome Thermodynamic Analysis Reduces siRNA Off-Target Effects

    PubMed Central

    Chen, Xi; Liu, Peng; Chou, Hui-Hsien

    2013-01-01

    Small interfering RNAs (siRNAs) are important tools for knocking down targeted genes, and have been widely applied to biological and biomedical research. To design siRNAs, two important aspects must be considered: the potency in knocking down target genes and the off-target effect on any nontarget genes. Although many studies have produced useful tools to design potent siRNAs, off-target prevention has mostly been delegated to sequence-level alignment tools such as BLAST. We hypothesize that whole-genome thermodynamic analysis can identify potential off-targets with higher precision and help us avoid siRNAs that may have strong off-target effects. To validate this hypothesis, two siRNA sets were designed to target three human genes IDH1, ITPR2 and TRIM28. They were selected from the output of two popular siRNA design tools, siDirect and siDesign. Both siRNA design tools have incorporated sequence-level screening to avoid off-targets, thus their output is believed to be optimal. However, one of the sets we tested has off-target genes predicted by Picky, a whole-genome thermodynamic analysis tool. Picky can identify off-target genes that may hybridize to a siRNA within a user-specified melting temperature range. Our experiments validated that some off-target genes predicted by Picky can indeed be inhibited by siRNAs. Similar experiments were performed using commercially available siRNAs and a few off-target genes were also found to be inhibited as predicted by Picky. In summary, we demonstrate that whole-genome thermodynamic analysis can identify off-target genes that are missed in sequence-level screening. Because Picky prediction is deterministic according to thermodynamics, if a siRNA candidate has no Picky predicted off-targets, it is unlikely to cause off-target effects. Therefore, we recommend including Picky as an additional screening step in siRNA design. PMID:23484018