Science.gov

Sample records for multiple organophosphorylated sites

  1. Mass spectrometry identifies multiple organophosphorylated sites on tubulin

    SciTech Connect

    Grigoryan, Hasmik Schopfer, Lawrence M. Peeples, Eric S. Duysen, Ellen G. Grigoryan, Marine Thompson, Charles M. Lockridge, Oksana

    2009-10-15

    Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 {sup o}C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.

  2. Mass spectrometry identifies multiple organophosphorylated sites on tubulin

    PubMed Central

    Grigoryan, Hasmik; Schopfer, Lawrence M.; Peeples, Eric S.; Duysen, Ellen G.; Grigoryan, Marine; Thompson, Charles M.; Lockridge, Oksana

    2009-01-01

    Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01–0.5 mM chlorpyrifos oxon for 24 h at 37 °C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals. PMID:19632257

  3. New Inhibitors of the Peripheral Site in Acetycholinesterase that Specifically Block Organophosphorylation

    DTIC Science & Technology

    1999-09-01

    nonequilibrium analysis and steric blockade model for AChE inhibition by small peripheral site ligands. Additionally, we have examined the effect of known AChE...inactivation of AChE. 14. SUBJECT TERMS 15. NUMBER OF PAGES acetylcholinesterase, organophosphates, inhibitors, nonequilibrium 61 analysis , combinatorial...was both experimentally and computationally confirmed for the first time in the AChE field by our laboratory. The analysis led us further to propose

  4. New Inhibitors of the Peripheral Site in Acetylcholinesterase that Specifically Block Organophosphorylation

    DTIC Science & Technology

    2000-09-01

    C in buffer, in most cases under pseudo first- que, Departament de Fisicoquimica , Universitat de Barcelona) and order conditions in which the... Fisicoquimica , consistent with thermodynamic data showing a reduction of Universitat de Barcelona, for providing atomic coordinates of only 5- to 7-fold in

  5. Multiple Scatters in Single Site Gamma Backgrounds

    SciTech Connect

    Brodsky, J. P.

    2016-09-16

    nEXO aims to reduce its gamma backgrounds by taking advantage of the fact that a large number of gammas that would otherwise be backgrounds will undergo multiple compton scattering in the TPC and produce spatially distinct signals. These multi-sited (MS) events can be excluded from the 0νββ search.

  6. Software Portability Considerations for Multiple Applications over Multiple Sites

    PubMed Central

    Munnecke, Thomas

    1981-01-01

    There are great benefits to be obtained by distributing the cost of software development over multiple sites. Both economies and dis-economies of scale become prominent when broad-based software portability is looked at carefully. However, traditional data processing techniques are oriented to making specific users, rather than general software for a class of users. This trend toward overspecification is getting worse with traditional data processing languages, while other standard languages are confronting it.

  7. Multiple Proteases to Localize Oxidation Sites

    PubMed Central

    Gu, Liqing; Robinson, Renã A. S.

    2015-01-01

    Proteins present in cellular environments with high levels of reactive oxygen and nitrogen species and/or low levels of antioxidants are highly susceptible to oxidative post-translational modification (PTM). Irreversible oxidative PTMs can generate a complex distribution of modified protein molecules, recently termed as proteoforms. Using ubiquitin as a model system, we mapped oxidative modification sites using trypsin, Lys-C, and Glu-C peptides. Several M+16 Da proteoforms were detected as well as proteoforms that include other previously unidentified oxidative modifications. This work highlights the use of multiple protease digestions to give insights to the complexity of oxidative modifications possible in bottom-up analyses. PMID:25775238

  8. Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: A biomarker of exposure to organophosphate agents

    SciTech Connect

    Wang, Liming; Du, Dan; Lu, Donglai; Lin, Chiann Tso; Smith, Jordan N.; Timchalk, Charles; Liu, Fengquan; Wang, Jun; Lin, Yuehe

    2011-05-05

    A sandwich enzyme-linked immunosorbent assay (sELISA) is developed for detection of organophosphorylated butyrylcholinesterase (OP-BChE), a potential biomarker for human exposure to organophosphate insecticides and nerve agents. A pair of antibodies specific to OP-BChE adduct were identified through systematic screening of several anti BChE antibodies (anti-BChE) and anti-phosphoserine antibodies (anti-Pser) from different sources. The selected anti-BChE (set as capture antibody) antibodies recognize both phosphorylated and nonphosphorylated BChE. These antibodies can therefore be used to capture both BChE and OP-BChE from the sample matrices. The anti- Pser (set as detecting antibody) was used to recognize the OP moiety of OP-BChE adducts. With the combination of the selected antibody pair, several key parameters (such as the concentration of anti-BChE and anti-Pser, and the blocking agent) were optimized to enhance the sensitivity and selectivity of the sELISA. Under the optimal conditions, the sELISA has shown a wide linear range from 0.03 nM to 30 nM, with a detection limit of 0.03 nM. Furthermore, the sELISA was successfully applied to detect OP-BChE using in-vitro biological samples such as rat plasma spiked with OP-BChE with excellent adduct recovery (z>99 %). These results demonstrate that this novel approach holds great promise to develop an ELISA kit and offers a simple and cost-effective tool for screening/evaluating exposure to organophosphate insecticides and nerve agents.

  9. HPV Vaccine Effective at Multiple Anatomic Sites

    Cancer.gov

    A new study from NCI researchers finds that the HPV vaccine protects young women from infection with high-risk HPV types at the three primary anatomic sites where persistent HPV infections can cause cancer. The multi-site protection also was observed at l

  10. Conducting multiple-site clinical trials in medical rehabilitation research.

    PubMed

    Fuhrer, Marcus J

    2005-11-01

    This article examines the distinctive opportunities and challenges of multiple-site clinical trials, as distinguished from trials conducted by a single program. Among the topics discussed are: the role of clinical trials generally in medical rehabilitation research; definitions of "clinical trials" and "multiple site"; the distinction between exploratory and capstone trials; the potential advantages and disadvantages of multiple-site trials; planning issues in terms of sampling designs, the choice of outcome measures, approaches to dealing with intervention fidelity, organizational structures, authorship, and the conduct of collateral studies; and implementation issues in terms of pilot-testing procedures, handling emerging issues, and optimizing communication.

  11. Extending a clinical repository to include multiple sites.

    PubMed Central

    Marrs, K. A.; Kahn, M. G.

    1995-01-01

    With the consolidation of health care organizations and services, a clinical repository comprising data from a single site is no longer sufficient. Individual patient data are now spread across multiple sites comprising a single enterprise. Users require an integrated view, or at least a common view, of these clinical data across multiple sites. Many issues arise when one tries to merge data from multiple, distinct organizations into an existing schema. We have addressed these issues while extending our clinical repository for Barnes Hospital with data from Jewish Hospital, both of which are members of the recently formed BJC Health System. We describe the architecture of our existing repository, approaches and issues in extending this repository to include multiple sites, and the specific issues we addressed in our system. PMID:8563308

  12. Identifying Martian Hydrothermal Sites: Geological Investigation Utilizing Multiple Datasets

    NASA Technical Reports Server (NTRS)

    Dohm, J. M.; Baker, V. R.; Anderson, R. C.; Scott, D. H.; Rice, J. W., Jr.; Hare, T. M.

    2000-01-01

    Comprehensive geological investigations of martian landscapes that may have been modified by magmatic-driven hydrothermal activity, utilizing multiple datasets, will yield prime target sites for future hydrological, mineralogical, and biological investigations.

  13. Double clicking for site-specific coupling of multiple enzymes.

    PubMed

    Lim, Sung In; Cho, Jinhwan; Kwon, Inchan

    2015-09-14

    A method to site-specifically couple multiple enzymes is reported. The approach is based on the site-specific incorporation of a clickable non-natural amino acid into enzymes and two compatible click reactions. The multi-enzyme reaction system exhibited enhanced catalytic efficiency over the respective free enzymes.

  14. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  15. Modular risk analysis for assessing multiple waste sites

    SciTech Connect

    Whelan, G.; Buck, J.W.; Nazarali, A.

    1994-06-01

    Human-health impacts, especially to the surrounding public, are extremely difficult to assess at installations that contain multiple waste sites and a variety of mixed-waste constituents (e.g., organic, inorganic, and radioactive). These assessments must address different constituents, multiple waste sites, multiple release patterns, different transport pathways (i.e., groundwater, surface water, air, and overland soil), different receptor types and locations, various times of interest, population distributions, land-use patterns, baseline assessments, a variety of exposure scenarios, etc. Although the process is complex, two of the most important difficulties to overcome are associated with (1) establishing an approach that allows for modifying the source term, transport, or exposure component as an individual module without having to re-evaluate the entire installation-wide assessment (i.e., all modules simultaneously), and (2) displaying and communicating the results in an understandable and useable maimer to interested parties. An integrated, physics-based, compartmentalized approach, which is coupled to a Geographical Information System (GIS), captures the regional health impacts associated with multiple waste sites (e.g., hundreds to thousands of waste sites) at locations within and surrounding the installation. Utilizing a modular/GIS-based approach overcomes difficulties in (1) analyzing a wide variety of scenarios for multiple waste sites, and (2) communicating results from a complex human-health-impact analysis by capturing the essence of the assessment in a relatively elegant manner, so the meaning of the results can be quickly conveyed to all who review them.

  16. Using Multiple Unmanned Systems for a Site Security Task

    SciTech Connect

    Matthew O. Anderson; Curtis W. Nielsen; Mark D. McKay; Derek C. Wadsworth; Ryan C. Hruska; John A. Koudelka

    2009-04-01

    Unmanned systems are often used to augment the ability of humans to perform challenging tasks. While the value of individual unmanned vehicles have been proven for a variety of tasks, it is less understood how multiple unmanned systems should be used together to accomplish larger missions such as site security. The purpose of this paper is to discuss efforts by researchers at the Idaho National Laboratory (INL) to explore the utility and practicality of operating multiple unmanned systems for a site security mission. This paper reviews the technology developed for a multi-agent mission and summarizes the lessons-learned from a technology demonstration.

  17. The immune system--multiple sites but one system.

    PubMed

    Harleman, Johannes H

    2006-07-01

    Recently several guidelines were published on immunotoxicity. Validation studies have shown that detailed extended examination of the immune system is able to flag immunotoxic compounds. Parameters of the examination are presented. In the final examination it is important that the whole immune system is evaluated as one functional system--multiple sites but one system.

  18. Fatal cryptococcosis involving multiple sites in an immunocompetent child.

    PubMed

    Kaur, H; Zaman, K; Thapa, B R; Rudramurthy, S M

    2015-02-01

    Disseminated cryptococcosis is less common in immunocompetent individuals. Herein, we report a fatal case of cryptococcosis in apparently immunocompetent child with multiple site involvement. The yeast isolated from cerebrospinal fluid, blood, endotracheal, gastric and lymph node aspirate was identified by molecular method as Cryptococcus neoformans var. grubii.

  19. EPA True NO2 ground site measurements ?? multiple sites, TCEQ ground site measurements of meteorological and air pollution parameters ?? multiple sites ,GeoTASO NO2 Vertical Column

    EPA Pesticide Factsheets

    EPA True NO2 ground site measurements ?? multiple sites - http://www-air.larc.nasa.gov/cgi-bin/ArcView/discover-aq.tx-2013; TCEQ ground site measurements of meteorological and air pollution parameters ?? multiple sites - http://www-air.larc.nasa.gov/cgi-bin/ArcView/discover-aq.tx-2013; GeoTASO NO2 Vertical Column - http://www-air.larc.nasa.gov/cgi-bin/ArcView/discover-aq.tx-2013?FALCON=1This dataset is associated with the following publication:Nowlan, C., X. Lu, J. Leitch, K. Chance, G. González Abad, C. Lu, P. Zoogman, J. Cole, T. Delker, W. Good, F. Murcray, L. Ruppert, D. Soo, M. Follette-Cook, S. Janz, M. Kowalewski, C. Loughner, K. Pickering, J. Herman, M. Beaver, R. Long, J. Szykman, L. Judd, P. Kelley, W. Luke, X. Ren, and J. Al-Saadi. Nitrogen dioxide observations from the Geostationary Trace gas and Aerosol Sensor Optimization (GeoTASO) airborne instrument: Retrieval algorithm and measurements during DISCOVER-AQ Texas 2013. Atmospheric Measurement Techniques. Copernicus Publications, Katlenburg-Lindau, GERMANY, 9(6): 2647-2668, (2016).

  20. Hereditary Angioedema Attacks: Local Swelling at Multiple Sites.

    PubMed

    Hofman, Zonne L M; Relan, Anurag; Hack, C Erik

    2016-02-01

    Hereditary angioedema (HAE) patients experience recurrent local swelling in various parts of the body including painful swelling of the intestine and life-threatening laryngeal oedema. Most HAE literature is about attacks located in one anatomical site, though it is mentioned that HAE attacks may also involve multiple anatomical sites simultaneously. A detailed description of such multi-location attacks is currently lacking. This study investigated the occurrence, severity and clinical course of HAE attacks with multiple anatomical locations. HAE patients included in a clinical database of recombinant human C1-inhibitor (rhC1INH) studies were evaluated. Visual analog scale scores filled out by the patients for various symptoms at various locations and investigator symptoms scores during the attack were analysed. Data of 219 eligible attacks in 119 patients was analysed. Thirty-three patients (28%) had symptoms at multiple locations in anatomically unrelated regions at the same time during their first attack. Up to five simultaneously affected locations were reported. The observation that severe HAE attacks often affect multiple sites in the body suggests that HAE symptoms result from a systemic rather than from a local process as is currently believed.

  1. Multiple-site estimations in probabilistic seismic hazard assessment

    NASA Astrophysics Data System (ADS)

    Sokolov, Vladimir; Ismail-Zadeh, Alik

    2016-04-01

    We analyze specific features of multiple-site probabilistic seismic hazard assessment (PSHA), i.e. annual rate of ground motion level exceedance in at least one site of several sites of interest located within in an area or along a linear extended object. The relation between the multiple-scale hazard estimations and strong ground-motion records obtained during the 2008 Wenchuan (China) Mw 7.9 earthquake is discussed. The ground-motion records may be considered as an example of ground motion exceeding the design level estimated using the classical point-wise PSHA. We showed that the multiple-site hazard (MSH) assessment, when being performed for standard return period 475 years, provide reasonable estimations of the ground motions that may occur during the earthquake, parameters of which are close to maximum possible events accepted in PSHA for the region. Thus the MSH may be useful in estimation of maximum considered earthquake ground motion for the considered territory taking into account its extent.

  2. Prioritization of multiple CERCLA sites using the analytic hierarchy process

    SciTech Connect

    Brown, G.M.; Olis, A.; Georgariou, P.N.

    1994-12-31

    This paper presents an innovative technique, the Analytic Hierarchy Process (AHP), that was used to prioritize multiple potential hazardous waste sites at a large Department of Defense (DoD) facility identified on the Superfund`s National Priorities List (NPL). Most DoD facilities listed on the NPL are involved in complex investigations and cleanup activities that last for years and cost millions of dollars. Large facilities commonly have dozens of potentially contaminated sites. The AHP was developed to assist people in integrating qualitative and quantitative decision-making. This versatile mathematical technique has since been used for such diverse purposes as making capital investment decisions in third world economies and choosing between alternative wastewater treatment technologies. In this paper, the authors will demonstrate how the AHP can be used in hazardous waste site prioritization where dozens of individuals sites have to be investigated with limited resources, general lack of qualitative and quantitative data, and conflicting priorities.

  3. Creating BHb-imprinted magnetic nanoparticles with multiple binding sites.

    PubMed

    Li, Yanxia; Chen, Yiting; Huang, Lu; Lou, BenYong; Chen, Guonan

    2017-01-16

    A kind of protein imprinted over magnetic Fe3O4@Au multifunctional nanoparticles (NPs) with multiple binding sites was synthesized and investigated. Magnetic Fe3O4@Au NPs as carrier materials were modified with 4-mercaptophenylboronic acid (MPBA) and mercaptopropionic acid (MPA) to introduce boronic acids and carboxyl groups. Using Bovine Hemoglobin (BHb) as a template, a polydopamine(PDA)-based molecular imprinted film was fabricated to produce a kind of magnetic molecularly imprinted nanoparticle (MMIP), possessing multiple binding sites with benzene-diol, amino groups, boronic acids and carboxyl groups. The MMIPs exhibited an excellent imprinting effect and adsorption capacity (89.65± 0.38 mg g(-1)) toward the template protein. The results show that the MMIPs reached saturated adsorption at 0.5 mg mL(-1) within 90 min. The synthesized MMIPs are suitable for the removal and enrichment of the template protein in proteomics. The strategy of multiple binding sites paves the way for the preparation of functional nanomaterials in molecular imprinting techniques.

  4. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  5. HANFORD SITE WELDING PROGRAM SUCCESSFULLY PROVIDING A SINGLE SITE FUNCTION FOR USE BY MULTIPLE CONTRACTORS

    SciTech Connect

    CANNELL GR

    2009-11-19

    The Department of Energy, Richland Operations (DOE-RL) recently restructured its Hanford work scope, awarding two new contracts over the past several months for a total of three contracts to manage the sites cleanup efforts. DOE-RL met with key contractor personnel prior to and during contract transition to ensure site welding activities had appropriate oversight and maintained code compliance. The transition also provided an opportunity to establish a single site-wide function that would provide welding and materials engineering services to the Hanford site contractors: CH2M HILL Plateau Remediation Company (CHPRC); Mission Support Alliance (MSA); Washington River Protection Solutions (WRPS); and Washington Closure Hanford (WCH). Over the years, multiple and separate welding programs (amongst the several contractors) existed at the Hanford site leading to inefficiencies resulting from duplication of administrative efforts, maintenance of welding procedures, welder performance certifications, etc. The new, single program eliminates these inefficiencies. The new program, co-managed by two of the sites' new contractors, the CHPRC ('owner' of the program and responsible for construction welding services) and the MSA (provides maintenance welding services), provides more than just the traditional construction and maintenance welding services. Also provided, are welding engineering, specialty welding development/qualification for the closure of radioactive materials containers and materials evaluation/failure analysis. The following describes the new Hanford site welding program.

  6. Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round.

    PubMed

    Seyfang, Andreas; Jin, Jean Huaqian

    2004-01-15

    Site-directed mutagenesis is a powerful tool to explore the structure-function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins, and alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency. Here we describe the development of a simple and rapid method for site-directed mutagenesis of more than 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers with a unique 25-nucleotide tail each that are simultaneously annealed to template DNA together with the set of mutagenic primers in between. Following synthesis of the mutant strand by primer extension and ligation with T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction. We have employed this novel method to mutate simultaneously all 9 and 11 CUG leucine codons of the Hyg and Neo resistance genes, respectively, to the Candida albicans-friendly UUG leucine codon at 100% efficiency.

  7. Multiple-site-damage fatigue of riveted joints

    NASA Astrophysics Data System (ADS)

    Schijve, J.

    1992-04-01

    Results of fatigue tests in riveted lap joints, including fractographic observations, are presented and analyzed. They indicate that the load transmission in a riveted lap joint is a rather complex phenomenon, as confirmed by quite different crack initiation mechanisms. It may not be expected that fracture mechanics predicts early crack growth with a practically useful degree of accuracy. Most important, the fractographic analysis reveals, that if there are visible cracks in a riveted lap joint, there are many small cracks at most rivet holes in the same critical row of rivets. A multiple site damage situation is present. The conclusion also applies to fuselage lap joints. Scatter in laboratory tests in riveted lap joint specimens is relatively low. However, scatter of riveted joints in a fleet of aircraft may be larger for several reasons. Crack growth in riveted lap joints may be sufficiently slow to allow a timely detection in service. For aging aircraft this should be confirmed by full scale testing.

  8. Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression

    NASA Astrophysics Data System (ADS)

    Lengyel, Iván M.; Morelli, Luis G.

    2017-04-01

    Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

  9. Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression.

    PubMed

    Lengyel, Iván M; Morelli, Luis G

    2017-04-01

    Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

  10. Residual Strength Prediction of Fuselage Structures with Multiple Site Damage

    NASA Technical Reports Server (NTRS)

    Chen, Chuin-Shan; Wawrzynek, Paul A.; Ingraffea, Anthony R.

    1999-01-01

    This paper summarizes recent results on simulating full-scale pressure tests of wide body, lap-jointed fuselage panels with multiple site damage (MSD). The crack tip opening angle (CTOA) fracture criterion and the FRANC3D/STAGS software program were used to analyze stable crack growth under conditions of general yielding. The link-up of multiple cracks and residual strength of damaged structures were predicted. Elastic-plastic finite element analysis based on the von Mises yield criterion and incremental flow theory with small strain assumption was used. A global-local modeling procedure was employed in the numerical analyses. Stress distributions from the numerical simulations are compared with strain gage measurements. Analysis results show that accurate representation of the load transfer through the rivets is crucial for the model to predict the stress distribution accurately. Predicted crack growth and residual strength are compared with test data. Observed and predicted results both indicate that the occurrence of small MSD cracks substantially reduces the residual strength. Modeling fatigue closure is essential to capture the fracture behavior during the early stable crack growth. Breakage of a tear strap can have a major influence on residual strength prediction.

  11. Triaziflam and Diaminotriazine derivatives affect enantioselectively multiple herbicide target sites.

    PubMed

    Grossmann, K; Tresch, S; Plath, P

    2001-01-01

    Enantiomers of triaziflam and structurally related diaminotriazines were synthesized and their herbicidal mode of action was investigated. The compounds caused light and dark-dependent effects in multiple test systems including heterotrophic cleaver and photoautotrophic algal cell suspensions, the Hill reaction of isolated thylakoids and germinating cress seeds. Dose-response experiments revealed that the (S)-enantiomers of the compounds preferentially inhibited photosystem II electron transport (PET) and algae growth with efficacies similar to that of the herbicide atrazine. In contrast, the (R)-enantiomers of the diaminotriazines were up to 100 times more potent inhibitors of growth in cleaver cell suspensions and cress seedlings in the dark than the (S)-enantiomers. The most active compound, the (R)-enantiomer of triaziflam, inhibited shoot and root elongation of cress and maize seedlings at concentrations below 1 microM. The meristematic root tips swelled into a club shape which is typical for the action of mitotic disrupter herbicides and cellulose biosynthesis inhibitors. Microscopic examination using histochemical techniques revealed that triaziflam (R)-enantiomer blocks cell division in maize root tips 4 h after treatment. The chromosomes proceeded to a condensed state of prometaphase but were unable to progress further in the mitotic cycle. Disruption of mitosis was accompanied by a loss of spindle and phragmoplast micotubule arrays. Concomitantly, cortical microtubules decreased which could lead to isodiametric cell growth and consequently to root swelling. In addition, a decline in cellulose deposition in cell walls was found 24 h after treatment. Compared to the (R)-form, triaziflam (S)-enantiomer was clearly less active. The results suggest that triaziflam and related diaminotriazines affect enantioselectively multiple sites of action which include PET inhibitory activity, mitotic disruption by inhibiting microtubule formation and inhibition of

  12. A probabilistic fatigue analysis of multiple site damage

    NASA Technical Reports Server (NTRS)

    Rohrbaugh, S. M.; Ruff, D.; Hillberry, B. M.; Mccabe, G.; Grandt, A. F., Jr.

    1994-01-01

    The variability in initial crack size and fatigue crack growth is incorporated in a probabilistic model that is used to predict the fatigue lives for unstiffened aluminum alloy panels containing multiple site damage (MSD). The uncertainty of the damage in the MSD panel is represented by a distribution of fatigue crack lengths that are analytically derived from equivalent initial flaw sizes. The variability in fatigue crack growth rate is characterized by stochastic descriptions of crack growth parameters for a modified Paris crack growth law. A Monte-Carlo simulation explicitly describes the MSD panel by randomly selecting values from the stochastic variables and then grows the MSD cracks with a deterministic fatigue model until the panel fails. Different simulations investigate the influences of the fatigue variability on the distributions of remaining fatigue lives. Six cases that consider fixed and variable conditions of initial crack size and fatigue crack growth rate are examined. The crack size distribution exhibited a dominant effect on the remaining fatigue life distribution, and the variable crack growth rate exhibited a lesser effect on the distribution. In addition, the probabilistic model predicted that only a small percentage of the life remains after a lead crack develops in the MSD panel.

  13. A probabilistic fatigue analysis of multiple site damage

    NASA Astrophysics Data System (ADS)

    Rohrbaugh, S. M.; Ruff, D.; Hillberry, B. M.; McCabe, G.; Grandt, A. F., Jr.

    1994-09-01

    The variability in initial crack size and fatigue crack growth is incorporated in a probabilistic model that is used to predict the fatigue lives for unstiffened aluminum alloy panels containing multiple site damage (MSD). The uncertainty of the damage in the MSD panel is represented by a distribution of fatigue crack lengths that are analytically derived from equivalent initial flaw sizes. The variability in fatigue crack growth rate is characterized by stochastic descriptions of crack growth parameters for a modified Paris crack growth law. A Monte-Carlo simulation explicitly describes the MSD panel by randomly selecting values from the stochastic variables and then grows the MSD cracks with a deterministic fatigue model until the panel fails. Different simulations investigate the influences of the fatigue variability on the distributions of remaining fatigue lives. Six cases that consider fixed and variable conditions of initial crack size and fatigue crack growth rate are examined. The crack size distribution exhibited a dominant effect on the remaining fatigue life distribution, and the variable crack growth rate exhibited a lesser effect on the distribution. In addition, the probabilistic model predicted that only a small percentage of the life remains after a lead crack develops in the MSD panel.

  14. Bayesian reliability analysis in connection with the evaluation of multiple-site damage

    NASA Astrophysics Data System (ADS)

    Itagaki, Hiroshi; Asada, H.; Ito, S.

    A rivet splice model for analyzing multiple-site damage is presented. The effectiveness of the periodic inspection of multiple-site fatigue cracks along a rivet joint is discussed. The relative frequency distribution of visible crack length and joint posterior probability density are described.

  15. The Hanford Site multiple function badge demonstration project

    SciTech Connect

    Nelson, R.A.

    1993-03-01

    The US Department of Energy Hanford Site is an original defense production site dating from World War II. The US Department of Energy assigned a new waste management mission for the Hanford Site. This mission requires fewer manned security posts and more areas restricted because of safety and environmental concerns. Entry into these areas requires specific safety and environmental qualifications, which could be controlled by automated access control systems. Automated entry reduces costs of managing the Site but requires health physics, safety, employee training, security databases integration, and a network system. Another approach, avoiding integration, is individuals carrying this information on their person as a picture identity badge and access control token. The health physics, safety, employee training, and security regulations that define access requirements may be best served using a smart card as the access control token. This paper discusses the need for information distribution and automated access control, potential approaches for solving the need, and smart card work at the Hanford Site.

  16. Risk Prediction Models for Other Cancers or Multiple Sites

    Cancer.gov

    Developing statistical models that estimate the probability of developing other multiple cancers over a defined period of time will help clinicians identify individuals at higher risk of specific cancers, allowing for earlier or more frequent screening and counseling of behavioral changes to decrease risk.

  17. EVALUATING NATURAL BIODEGRADATION OF MTBE AT MULTIPLE UST SITES

    EPA Science Inventory

    Until very recently, methyl t-butyl ether (MTBE) was considered non-biodegradable in the subsurface. This has been an impediment in applying remediation by natural attenuation (RNA) as a remedial strategy at MTBE-impacted sites. Although a number of recent studies have demonst...

  18. EVALUATING NATURAL BIODEGRADATION OF MTBE AT MULTIPLE UST SITES

    EPA Science Inventory

    Until very recently, methyl t-butyl ether (MTBE) was considered non-biodegradable in the subsurface. This has been an impediment in applying remediation by natural attenuation (RNA) as a remedial strategy at MTBE-impacted sites. Although a number of recent studies have demonst...

  19. Reaching Site Closure for Groundwater under Multiple Regulatory Agencies

    SciTech Connect

    Glucksberg, N.; Shephard, Gene; Peters, Jay; Couture, B.

    2008-01-15

    Groundwater at the Connecticut Yankee Atomic Power Company (CYAPCO) Haddam Neck Plant (HNP) requires investigation of both radionuclides and chemical constituents in order to achieve closure. Cleanup criteria for groundwater are regulated both by federal and state agencies. These requirements vary in both numerical values as well as the duration of post remediation monitoring. The only consistent requirement is the development of a site conceptual model and an understanding of the hydrogeologic conditions that will govern contaminant transport and identify potential receptors. To successfully reach closure under each agency, it is paramount to understand the different requirements during the planning stages of the investigation. Therefore, the conceptual site model, groundwater transport mechanisms, and potential receptors must be defined. Once the hydrogeology is understood, a long term groundwater program can then be coordinated to meet each regulatory agency requirement to both terminate the NRC license and reach site closure under RCRA. Based on the different criteria, the CTDEP-LR (or RSR criteria) are not only bounding, but also requires the longest duration. As with most decommissioning efforts, regulatory attention is focused on the NRC, however, with the recent industry initiatives based on concern of tritium releases to groundwater at other plants, it is likely that the USEPA and state agencies may continue to drive site investigations. By recognizing these differences, data quality objectives can include all agency requirements, thus minimizing rework or duplicative efforts. CYAPCO intends to complete groundwater monitoring for the NRC and CTDEP-RD by July 2007. However, because shallow remediations are still being conducted, site closure under USEPA and CTDEP-LR is projected to be late 2011.

  20. Multiple missions: The 300 Area in Hanford Site history

    SciTech Connect

    Gerber, M.S.

    1993-09-01

    This report provides an historical overview of the role of the 300 Area buildings at the Hanford Reservation. Topics covered are: Early fuel fabrication at the Hanford site (313 and 314 Buildings); N reactor fuel fabrication in the 300 Area; 305 test pile was Hanford`s first operating reactor; Early process improvement chemical research (321 and 3706 Buildings); Major 1952 and 1953 expansions in the 300 area (325 and 329 Buildings); Early 300 area facilities constructed to support reactor development (326 and 327 Buildings); Hanford site ventures with the peaceful atom (309, 308 and 318 Buildings); Modern 300 Area Buildings; Significant miscellaneous buildings in the 300 area; 300 Area process waste handling and disposal.

  1. Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

    SciTech Connect

    Sloan, J.W.

    1984-01-01

    These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) have been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.

  2. Subcellular probes for neurochemical recording from multiple brain sites.

    PubMed

    Schwerdt, Helen N; Kim, Min Jung; Amemori, Satoko; Homma, Daigo; Yoshida, Tomoko; Shimazu, Hideki; Yerramreddy, Harshita; Karasan, Ekin; Langer, Robert; Graybiel, Ann M; Cima, Michael J

    2017-03-14

    Dysregulation of neurochemicals, in particular, dopamine, is epitomized in numerous debilitating disorders that impair normal movement and mood aspects of our everyday behavior. Neurochemical transmission is a neuron-specific process, and further exhibits region-specific signaling in the brain. Tools are needed to monitor the heterogeneous spatiotemporal dynamics of dopamine neurotransmission without compromising the physiological processes of the neuronal environment. We developed neurochemical probes that are ten times smaller than any existing dopamine sensor, based on the size of the entire implanted shaft and its sensing tip. The microfabricated probe occupies a spatial footprint (9 μm) coordinate with the average size of individual neuronal cells (∼10 μm). These cellular-scale probes were shown to reduce inflammatory response of the implanted brain tissue environment. The probes are further configured in the form of a microarray to permit electrochemical sampling of dopamine and other neurotransmitters at unprecedented spatial densities and distributions. Dopamine recording was performed concurrently from up to 16 sites in the striatum of rats, revealing a remarkable spatiotemporal contrast in dopamine transmission as well as site-specific pharmacological modulation. Collectively, the reported platform endeavors to enable high density mapping of the chemical messengers fundamentally involved in neuronal communication through the use of minimally invasive probes that help preserve the neuronal viability of the implant environment.

  3. Reducing job coach assistance for supported workers with severe multiple disabilities: an alternative off-site/on-site model.

    PubMed

    Parsons, M B; Reid, D H; Green, C W; Browning, L B

    2001-01-01

    Adults with severe multiple disabilities constitute a very small percentage of individuals in supported work. When these persons do obtain community jobs, considerable assistance is usually required. We evaluated an off-site/on-site program for reducing job coach assistance provided for three adults with severe multiple disabilities in a part-time community job. Following observations of the supported workers' job performance in a publishing company, the job support reduction program was implemented while the individuals received more traditional day services when not at work. The program involved assessing the amount and type of assistance provided for each step in a worker's job tasks, and then reducing the assistance through environmental adaptations and instruction. After implementation in the nonwork setting, the adaptations and instruction were extended to the work site. Immediate reductions occurred in the amount of assistance provided by job coaches for each supported worker while on the job. No adverse effects on productivity were observed. These results suggest that an off-site/on-site approach to reducing work assistance represents a viable alternative to current supported work models. Social validity observations in 10 job sites highlighted the need to demonstrate ways to reduce work assistance provided for workers with severe multiple disabilities. Future research areas are noted, focusing on evaluating other models for enhancing supported work opportunities for people with highly significant disabilities.

  4. Reaching site closure for groundwater under multiple regulatory agencies

    SciTech Connect

    Glucksberg, N.; Couture, B.

    2007-07-01

    Groundwater at the Connecticut Yankee Atomic Power Company (CYAPCO) Haddam Neck Plant (HNP) has been impacted by both radionuclides and chemical constituents. Furthermore, the cleanup standards and closure requirements for HNP are regulated both by federal and state agencies. The only consistent requirement is the development of a site conceptual model and an understanding of the hydrogeologic conditions that will govern contaminant transport and identify potential receptors. The cleanup criteria to reach site closure for radionuclides is regulated by both the Nuclear Regulatory Commission (NRC) and the Connecticut Department of Environmental Protection (CTDEP) Bureau of Air Management, Radiological Division. For license termination under the NRC, the total effective dose equivalent (TEDE) for all media can not exceed 25 milli-Rem per year (mRem/yr) plus As Low as Reasonably Achievable (ALARA). The CTDEP has a similar requirement with the TEDE not to exceed 19 mRem/yr plus ALARA. To reach these criteria, derived concentration guideline levels (DCGLs) were developed for radiological exposures from three (3) media components; soil, existing groundwater and future groundwater from left-in place foundations or footings. Based on current conditions, the target dose contribution from existing and future groundwater is not to exceed 2 mRem/yr TEDE. After source (soil) remediation is complete, the NRC requires two (2) years of quarterly monitoring to demonstrate that groundwater quality meets the DCGLs and does not show an upward trend. CYAPCO's NRC License Termination Plan (LTP) specifies a minimum 18-month period of groundwater monitoring, as long as samples are collected during two spring/high water seasons, to verify the efficacy of remedial actions at HNP. In addition to the 19 mRem/yr criteria, the CTDEP also requires groundwater to be in compliance with the Remediation Standards Regulation (RSRs). There are no published criteria for radionuclides in the RSRs

  5. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  6. Malignant transformation of neurofibromas at multiple sites in a case of neurofibromatosis.

    PubMed Central

    Leslie, M. D.; Cheung, K. Y.

    1987-01-01

    A patient with classical neurofibromatosis is reported in whom malignant transformation of neurofibromas at multiple sites occurred, leading to a fatal outcome. One of these malignant tumours developed within the thyroid gland. Images Figure 1 Figure 2 PMID:3118347

  7. Effectiveness of multiple release sites in reintroduction of Persian fallow deer.

    PubMed

    Berger-Tal, Oded; Bar-David, Shirli; Saltz, David

    2012-02-01

    Releasing animals in more than one location may increase or decrease the probability of success of a reintroduction project, yet the question of how many release sites to use has received little attention. We used empirical data from the reintroduction program of the Persian fallow deer (Dama mesopotamica) (Galilee region in northern Israel) in an individual-based spatially explicit simulation model to assess the effects of releasing deer from multiple sites. We examined whether multiple release sites increase reintroduction success, and if so, whether the optimal number of sites for a given scenario can be determined and whether the outcome differs if animals are released alternately (i.e., the location of the release alternates yearly between sites) or consecutively (i.e., one release site is used for several years, then another is used, and so forth). We selected 8 potential release sites in addition to the original site and simulated the release of 180 individuals at a rate of 10 individuals per year in different combinations of the original site and 1-4 additional sites. In our model, releasing animals into the wild at multiple sites produced higher population growth and greater spatial expansion than releasing animals at only one site and a consecutive-release approach was superior to an alternate-release approach. We suggest that through the use of simulation modeling that is based on empirical data from previous releases, managers can make better-informed decisions regarding the use of multiple release sites and greatly improve the probability of reintroduction success.

  8. Multiple-site physician practices and their effect on service distribution.

    PubMed Central

    Cromley, E K; Albertsen, P C

    1993-01-01

    OBJECTIVE. This study explores the impact of multiple-site practices on the distribution of physician services within a medical service region. DATA SOURCES AND STUDY SETTING. A questionnaire was mailed to all urologists (100 percent response rate) practicing in north central Connecticut (the Hartford medical service area) and adjacent communities in September 1990. Data on community characteristics were obtained from the 1990 U.S. census and state government documents. STUDY DESIGN. Descriptive statistics and maps were used to summarize the attributes of single- and multiple-site practices and the communities where they were located. Key practice and community variables were analyzed. DATA COLLECTION/EXTRACTION METHODS. The questionnaires were coded and entered into a digital database with the tabulated community data. Responses of individual physicians were grouped by practice. PRINCIPAL FINDINGS. Multiple-site practices were common. Second-order sites accounted for 23 percent of total appointment capacity and were located in communities with higher than average elderly populations and incomes and lower than average minority populations. CONCLUSIONS. Analysis of multiple-site practices is important for the accurate assessment of medical service availability. Further research is needed to document the functioning of multiple-site practices across other specialties and geographic areas. PMID:8407340

  9. Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry.

    PubMed

    Zhang, Yanlin; Yu, Chuan-Yih; Song, Ehwang; Li, Shuai Cheng; Mechref, Yehia; Tang, Haixu; Liu, Xiaowen

    2015-12-04

    Glycosylation is one of the most common post-translational modifications in proteins, existing in ~50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites.

  10. One program, multiple training sites: does site of family medicine training influence professional practice location?

    PubMed

    Jamieson, Jean L; Kernahan, Jill; Calam, Betty; Sivertz, Kristin S

    2013-01-01

    Numerous strategies have been suggested to increase recruitment of family physicians to rural communities and smaller regional centers. One approach has been to implement distributed postgraduate education programs where trainees spend substantial time in such communities. The purpose of the current study was to compare the eventual practice location of family physicians who undertook their postgraduate training through a single university but who were based in either metropolitan or distributed, non-metropolitan communities. Since 1998, the Department of Family Practice at the University of British Columbia in Canada has conducted an annual survey of its residents at 2, 5, and 10 years after completion of training. The authors received Ethics Board approval to use this anonymized data to identify personal and educational factors that predict future practice location. The overall response rate was 45%. At 2 years (N=222), residents trained in distributed sites were 15 times more likely to enter practice in rural communities, small towns and regional centers than those who trained in metropolitan teaching centers. This was even more predictive for retention in non-urban practice sites. Among the subgroup of physicians who remained in a single practice location for more than a year preceding the survey, those who trained in smaller sites were 36 times more likely to choose a rural or regional practice setting. While the vast majority of those trained in metropolitan sites chose an urban practice location, a subgroup of those with some rural upbringing were more likely to practice in rural or regional settings. Trainees from distributed sites considered themselves more prepared for practice regardless of ultimate practice location. Participation in a distributed postgraduate family medicine training site is an important predictor of a non-urban practice location. This effect persists for 10 years after completion of training and is independent of other predictors of

  11. Atrial pacing at multiple sites in the Wolff-Parkinson-White syndrome.

    PubMed Central

    Denes, P; Wyndham, C R; Amat-y-Leon, F; Wu, D; Dhingra, R C; Miller, R H; Rosen, K M

    1977-01-01

    Atrial pacing at multiple sites was used in an attempt to predict the site of pre-excitation in 5 patients with Wolff-Parkinson-White syndrome with 5 different anomalous pathway locations (right anterior, right posterior, septal, left posterior, and left lateral). At least 3 atrial pacing sites were tested in each patient. Pacing sites tested included high right atrium, low lateral right atrium, low septal right atrium, proximal coronary sinus, and distal coronary sinus. Atrial stimulation sites with shortest and longest stimulus-delta intervals could be identified in each patient, the shortest stimulus-delta interval in each case ranging from 60 to 80 ms. The difference between the shortest and longest stimulus-delta interval in each case ranged from 60 to 110 ms. It was suggested that the site with the shortest stimulus-delta interval corresponded to a site close to the atrial insertion of the anomalous pathway. This hypothesis was confirmed in all cases (3 with epicardial mapping and 2 with retrograde atrial activation data). In conclusion, atrial pacing at multiple sites is helpful in predicting the site of anterogradely conducting anomalous pathways, and appears particularly useful for differentiation of right posterior, left posterior, and septal pre-excitation. Images PMID:861093

  12. Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms.

    PubMed

    Barber, Annika F; Carnevale, Vincenzo; Klein, Michael L; Eckenhoff, Roderic G; Covarrubias, Manuel

    2014-05-06

    Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na(+) (NaV) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial NaV channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.

  13. Electric properties and carrier multiplication in breakdown sites in multi-crystalline silicon solar cells

    SciTech Connect

    Schneemann, Matthias; Carius, Reinhard; Rau, Uwe; Kirchartz, Thomas

    2015-05-28

    This paper studies the effective electrical size and carrier multiplication of breakdown sites in multi-crystalline silicon solar cells. The local series resistance limits the current of each breakdown site and is thereby linearizing the current-voltage characteristic. This fact allows the estimation of the effective electrical diameters to be as low as 100 nm. Using a laser beam induced current (LBIC) measurement with a high spatial resolution, we find carrier multiplication factors on the order of 30 (Zener-type breakdown) and 100 (avalanche breakdown) as new lower limits. Hence, we prove that also the so-called Zener-type breakdown is followed by avalanche multiplication. We explain that previous measurements of the carrier multiplication using thermography yield results higher than unity, only if the spatial defect density is high enough, and the illumination intensity is lower than what was used for the LBIC method. The individual series resistances of the breakdown sites limit the current through these breakdown sites. Therefore, the measured multiplication factors depend on the applied voltage as well as on the injected photocurrent. Both dependencies are successfully simulated using a series-resistance-limited diode model.

  14. Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms

    PubMed Central

    Barber, Annika F.; Carnevale, Vincenzo; Klein, Michael L.; Eckenhoff, Roderic G.; Covarrubias, Manuel

    2014-01-01

    Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na+ (NaV) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial NaV channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action. PMID:24753583

  15. Case-control analysis in highway safety: Accounting for sites with multiple crashes.

    PubMed

    Gross, Frank

    2013-12-01

    There is an increased interest in the use of epidemiological methods in highway safety analysis. The case-control and cohort methods are commonly used in the epidemiological field to identify risk factors and quantify the risk or odds of disease given certain characteristics and factors related to an individual. This same concept can be applied to highway safety where the entity of interest is a roadway segment or intersection (rather than a person) and the risk factors of interest are the operational and geometric characteristics of a given roadway. One criticism of the use of these methods in highway safety is that they have not accounted for the difference between sites with single and multiple crashes. In the medical field, a disease either occurs or it does not; multiple occurrences are generally not an issue. In the highway safety field, it is necessary to evaluate the safety of a given site while accounting for multiple crashes. Otherwise, the analysis may underestimate the safety effects of a given factor. This paper explores the use of the case-control method in highway safety and two variations to account for sites with multiple crashes. Specifically, the paper presents two alternative methods for defining cases in a case-control study and compares the results in a case study. The first alternative defines a separate case for each crash in a given study period, thereby increasing the weight of the associated roadway characteristics in the analysis. The second alternative defines entire crash categories as cases (sites with one crash, sites with two crashes, etc.) and analyzes each group separately in comparison to sites with no crashes. The results are also compared to a "typical" case-control application, where the cases are simply defined as any entity that experiences at least one crash and controls are those entities without a crash in a given period. In a "typical" case-control design, the attributes associated with single-crash segments are weighted

  16. The benefits of a watershed approach to evaluating risks for multiple hazardous waste sites

    SciTech Connect

    Burris, J.A.; Pease, A.; Jop, K.; Connely, R.

    1994-12-31

    An integrated sampling program including chemical analyses of surface waters and sediments, measurement of water quality parameters, sampling of macroinvertebrate communities, and toxicity testing of soils and sediments was implemented on a US DOD Facility within three watersheds where multiple waste sites are located. The results of the study were used to identify the migration and distribution of contamination from individual waste sites, to identify any areas of obvious biological stress where future studies should be focused, to evaluate the need for sampling and analyses of aquatic organisms, to evaluate the influence of other non-point source and point sources of contamination of other stressors, and to identify sediment contamination in relation to the waste sites. This information was necessary to the design and implementation of ecological risk assessment for the individual waste sites. The methodology used to complete the study and its results will be presented. The focus of the discussion will be on the benefits of a watershed based sampling design (versus a more traditional site by site approach) in terms of cost savings, remedial decision making, and time savings. An additional discussion will focus on how this approach can be used to address the potential additive risks to a watershed resulting from the presence of multiple waste sites.

  17. Foothill yellow-legged frog (Rana boylii) oviposition site choice at multiple spatial scales

    Treesearch

    Amy J. Lind; Hartwell H. Welsh; Clara A. Wheeler

    2016-01-01

    Studies of resource selection at multiple scales are critical to understanding ecological and evolutionary attributes of a species. We analyzed relative abundance, habitat use, and oviposition site selection of Foothill Yellow-Legged Frogs (Rana boylii) at 11 localities across two geographic regions in California (northern Coast Range and Sierra...

  18. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (INTERSTATE LAND SALES REGISTRATION PROGRAM) LAND REGISTRATION General Requirements § 1710.15 Regulatory exemption—multiple site subdivision..., where the developer either owns contiguous land or holds an option or other evidence of intent...

  19. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (INTERSTATE LAND SALES REGISTRATION PROGRAM) LAND REGISTRATION General Requirements § 1710.15 Regulatory exemption—multiple site subdivision..., where the developer either owns contiguous land or holds an option or other evidence of intent...

  20. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (INTERSTATE LAND SALES REGISTRATION PROGRAM) LAND REGISTRATION General Requirements § 1710.15 Regulatory exemption—multiple site subdivision..., where the developer either owns contiguous land or holds an option or other evidence of intent...

  1. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (INTERSTATE LAND SALES REGISTRATION PROGRAM) LAND REGISTRATION General Requirements § 1710.15 Regulatory exemption—multiple site subdivision..., where the developer either owns contiguous land or holds an option or other evidence of intent...

  2. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (INTERSTATE LAND SALES REGISTRATION PROGRAM) LAND REGISTRATION General Requirements § 1710.15 Regulatory exemption—multiple site subdivision..., where the developer either owns contiguous land or holds an option or other evidence of intent...

  3. Managing data from multiple disciplines, scales, and sites to support synthesis and modeling

    USGS Publications Warehouse

    Olson, R. J.; Briggs, J. M.; Porter, J.H.; Mah, Grant R.; Stafford, S.G.

    1999-01-01

    The synthesis and modeling of ecological processes at multiple spatial and temporal scales involves bringing together and sharing data from numerous sources. This article describes a data and information system model that facilitates assembling, managing, and sharing diverse data from multiple disciplines, scales, and sites to support integrated ecological studies. Cross-site scientific-domain working groups coordinate the development of data associated with their particular scientific working group, including decisions about data requirements, data to be compiled, data formats, derived data products, and schedules across the sites. The Web-based data and information system consists of nodes for each working group plus a central node that provides data access, project information, data query, and other functionality. The approach incorporates scientists and computer experts in the working groups and provides incentives for individuals to submit documented data to the data and information system.

  4. Traffic Management Advisor: Iterative Field Development and Assessment at Multiple Sites

    NASA Technical Reports Server (NTRS)

    Sanford, Beverly D.; Lee, Katharine K.; Harwood, Kelly; Denery, Dallas G. (Technical Monitor)

    1995-01-01

    Previous studies have demonstrated the necessity of involving users in the development of automation aids, especially for complex domains such as air traffic control (ATC). Traditional development only demanded a single field test to validate a completed system, but a more iterative combination of development and assessment ensures that the technology meets the requirements of its application domain. Exposure across an adequate spectrum of field users is also required during development, and the use of multiple development sites provides an opportunity to consider individual facility cultures as they relate to implementation strategies. The development of the Center/TRACON Automation System (CTAS) Traffic Management Advisor (TMA) at the Denver and Dallas ATC facilities demonstrates successful iterative development and assessment at multiple field sites. The use of field development changes the nature of assessment. As development progresses, periodic assessments are required to validate that system development is progressing along an appropriate track. In the development of the TMA, assessments were performed based on software in the field, and input from traffic managers was analyzed and incorporated into subsequent releases of the TMA, to be reassessed in the field. This has led to a tool with operational suitability and broad user acceptance. Assessment at multiple sites provides a more generalizable perspective that allows the production of a system that is both generic enough to be used at different sites and tailored enough to be of use at any site. In addition to providing a better understanding of specific facility requirements, the use of multiple assessment sites in the development of TMA has provided an opportunity to consider individual facility operations, procedures and cultures as they relate to development and implementation strategies.

  5. Traffic Management Advisor: Iterative Field Development and Assessment at Multiple Sites

    NASA Technical Reports Server (NTRS)

    Sanford, Beverly D.; Lee, Katharine K.; Harwood, Kelly; Denery, Dallas G. (Technical Monitor)

    1995-01-01

    Previous studies have demonstrated the necessity of involving users in the development of automation aids, especially for complex domains such as air traffic control (ATC). Traditional development only demanded a single field test to validate a completed system, but a more iterative combination of development and assessment ensures that the technology meets the requirements of its application domain. Exposure across an adequate spectrum of field users is also required during development, and the use of multiple development sites provides an opportunity to consider individual facility cultures as they relate to implementation strategies. The development of the Center/TRACON Automation System (CTAS) Traffic Management Advisor (TMA) at the Denver and Dallas ATC facilities demonstrates successful iterative development and assessment at multiple field sites. The use of field development changes the nature of assessment. As development progresses, periodic assessments are required to validate that system development is progressing along an appropriate track. In the development of the TMA, assessments were performed based on software in the field, and input from traffic managers was analyzed and incorporated into subsequent releases of the TMA, to be reassessed in the field. This has led to a tool with operational suitability and broad user acceptance. Assessment at multiple sites provides a more generalizable perspective that allows the production of a system that is both generic enough to be used at different sites and tailored enough to be of use at any site. In addition to providing a better understanding of specific facility requirements, the use of multiple assessment sites in the development of TMA has provided an opportunity to consider individual facility operations, procedures and cultures as they relate to development and implementation strategies.

  6. Multiple-regression equations for estimating low flows at ungaged stream sites in Ohio

    USGS Publications Warehouse

    Koltun, G.F.; Schwartz, R.R.

    1987-01-01

    This report presents multiple-regression equations for estimating selected low-flow characteristics for most unregulated Ohio streams at sites where little or no discharge data are available. The equations relate combinations of drainage area, main-channel length, main-channel slope, average basin elevation, forested area, average annual precipitation, and an index of infiltration to low flows with durations of 7 and 30 days and average recurrence intervals of 2 and 10 years. Data from 132 long-term continuous-record gaging stations and partial-record sites in Ohio were used in the analyses. Multiple-regression analyses were first performed by using data from all 132 sites in an attempt to develop equations that would be applicable statewide. Standard errors for the statewide equations were too high (111 to 189 percent) for them to be of practical use in estimating low streamflows. Data for the state were then subdivided into five regions, and multiple-regression equations were developed for each region. Standard errors for four of the five regions improved, and raged from 43 to 106 percent. Standard errors for region 5 remained high (74 to 129 percent). The multiple-regression equations presented in this report are not applicable to streams with significant low-flow regulation. The equations also are not applicable if (1) the site has been gaged and low-flow estimates have been developed from gaging-station records, (2) low flow can be estimated by the drainage-area transference method from data for a nearby gaged site, or (3) a sufficient number of partial-record measurements made at the site can be adquately correlated with concurrent base flows at a suitable index station.

  7. MBSTAR: multiple instance learning for predicting specific functional binding sites in microRNA targets

    NASA Astrophysics Data System (ADS)

    Bandyopadhyay, Sanghamitra; Ghosh, Dip; Mitra, Ramkrishna; Zhao, Zhongming

    2015-01-01

    MicroRNA (miRNA) regulates gene expression by binding to specific sites in the 3'untranslated regions of its target genes. Machine learning based miRNA target prediction algorithms first extract a set of features from potential binding sites (PBSs) in the mRNA and then train a classifier to distinguish targets from non-targets. However, they do not consider whether the PBSs are functional or not, and consequently result in high false positive rates. This substantially affects the follow up functional validation by experiments. We present a novel machine learning based approach, MBSTAR (Multiple instance learning of Binding Sites of miRNA TARgets), for accurate prediction of true or functional miRNA binding sites. Multiple instance learning framework is adopted to handle the lack of information about the actual binding sites in the target mRNAs. Biologically validated 9531 interacting and 973 non-interacting miRNA-mRNA pairs are identified from Tarbase 6.0 and confirmed with PAR-CLIP dataset. It is found that MBSTAR achieves the highest number of binding sites overlapping with PAR-CLIP with maximum F-Score of 0.337. Compared to the other methods, MBSTAR also predicts target mRNAs with highest accuracy. The tool and genome wide predictions are available at http://www.isical.ac.in/~bioinfo_miu/MBStar30.htm.

  8. Discovery of multiple hidden allosteric sites by combining Markov state models and experiments.

    PubMed

    Bowman, Gregory R; Bolin, Eric R; Hart, Kathryn M; Maguire, Brendan C; Marqusee, Susan

    2015-03-03

    The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets--called hidden allosteric sites--remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond- to millisecond-timescale fluctuations of a protein's structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target--TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.

  9. Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus.

    PubMed

    Bryda, Elizabeth C; Pearson, Michael; Agca, Yuksel; Bauer, Beth A

    2006-12-01

    Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.

  10. Evaluation of French-language internet sites dealing with multiple sclerosis

    PubMed Central

    Maillart, Elisabeth; Gallice, Thibault; Allaf, Bashar

    2016-01-01

    Background Information available on the internet has changed patient–neurologist relationships. Its evaluation for multiple sclerosis is only partial, regardless of the language used. Objective We aim to evaluate the content quality and ranking indexes of French-language sites dealing with multiple sclerosis. Methods Two French terms and three search engines were used to identify the sites whose ranking indexes were calculated according to their positions on each page designated by the search engines. Three evaluators used the DISCERN questionnaire to assess the content quality of the 25 selected sites. The sites were classified according to the mean of the evaluators’ grades. Grading agreement between evaluators was calculated. Ranking indexes were computed as a rank/100. Results Content level was deemed mediocre, with poor referencing of the information provided. The naïve and two expert evaluators’ grades differed. Content quality disparity was found within the different website categories, except for institutional sites. No correlation was found between content quality and ranking index. Conclusion The information available was heterogeneous. Physicians should guide patients in their internet searches for information so that they can benefit from good-quality input which is potentially able to improve their management. PMID:28607728

  11. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing

    PubMed Central

    Malina, Abba; Cameron, Christopher J. F.; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

    2015-01-01

    In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification. PMID:26644285

  12. Calculating the habitable zones of multiple star systems with a new interactive Web site

    SciTech Connect

    Müller, Tobias W. A.; Haghighipour, Nader

    2014-02-10

    We have developed a comprehensive methodology and an interactive Web site for calculating the habitable zone (HZ) of multiple star systems. Using the concept of spectral weight factor, as introduced in our previous studies of the calculations of HZ in and around binary star systems, we calculate the contribution of each star (based on its spectral energy distribution) to the total flux received at the top of the atmosphere of an Earth-like planet, and use the models of the HZ of the Sun to determine the boundaries of the HZ in multiple star systems. Our interactive Web site for carrying out these calculations is publicly available at http://astro.twam.info/hz. We discuss the details of our methodology and present its application to some of the multiple star systems detected by the Kepler space telescope. We also present the instructions for using our interactive Web site, and demonstrate its capabilities by calculating the HZ for two interesting analytical solutions of the three-body problem.

  13. Multiple ligand docking by Glide: implications for virtual second-site screening

    NASA Astrophysics Data System (ADS)

    Vass, Márton; Tarcsay, Ákos; Keserű, György M.

    2012-07-01

    Performance of Glide was evaluated in a sequential multiple ligand docking paradigm predicting the binding modes of 129 protein-ligand complexes crystallized with clusters of 2-6 cooperative ligands. Three sampling protocols (single precision—SP, extra precision—XP, and SP without scaling ligand atom radii—SP hard) combined with three different scoring functions (GlideScore, Emodel and Glide Energy) were tested. The effects of ligand number, docking order and druglikeness of ligands and closeness of the binding site were investigated. On average 36 % of all structures were reproduced with RMSDs lower than 2 Å. Correctly docked structures reached 50 % when docking druglike ligands into closed binding sites by the SP hard protocol. Cooperative binding to metabolic and transport proteins can dramatically alter pharmacokinetic parameters of drugs. Analyzing the cytochrome P450 subset the SP hard protocol with Emodel ranking reproduced two-thirds of the structures well. Multiple ligand binding is also exploited by the fragment linking approach in lead discovery settings. The HSP90 subset from real life fragment optimization programs revealed that Glide is able to reproduce the positions of multiple bound fragments if conserved water molecules are considered. These case studies assess the utility of Glide in sequential multiple docking applications.

  14. A Single Primary Site Obstruction May Lead to Sleep-Disordered Breathing in Multiple Sites: An Animal Model.

    PubMed

    Lee, Myung-Chul; Rhee, Chae-Seo; Joe, Sam; Yoon, In-Young; Kim, Jeong-Whun

    2016-04-01

    This study aimed to investigate the dynamic upper airway changes occurring in an obstructive sleep apnea (OSA) rabbit model using dynamic computerized tomography (CT). In this study, 2.5 U of botulinum toxin type A was injected into the genioglossus to paralyze the tongue in the OSA group (n = 7). The control group was injected with normal saline (n = 7). Apnea-hypopnea index was measured using ApneaLink at baseline and at 1, 2, 3, 4, 6, and 8 weeks post-injection. Anterior to posterior (AP) and transverse diameters at the levels of the palate and tongue base were measured using a dynamic CT at baseline and at 1 and 2 weeks post-injection. The success rate of OSA induction was higher in the OSA group (P = .02). In the OSA group, transverse and AP diameters at the palate level and AP diameter at the tongue base level significantly decreased 2 weeks post-injection (P = .01). A single obstruction site may constitute the primary pathogenesis in some OSA patients, even when the involvement of multiple sites is indicated. These findings may contribute to the identification of OSA pathogenesis and improve the planning of treatment based on the primary cause of OSA. © The Author(s) 2015.

  15. Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata.

    PubMed

    Tobiason, D M; Lenich, A G; Glasgow, A C

    1999-04-02

    The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

  16. Early establishment of multiple release site connectivity between interneurons and pyramidal neurons in the developing hippocampus.

    PubMed

    Groc, Laurent; Gustafsson, Bengt; Hanse, Eric

    2003-05-01

    The strength of the synaptic transmission between two neurons critically depends on the number of release sites connecting the neurons. Here we examine the development of connectivity between gamma-aminobutyric acid (GABA)ergic interneurons and CA1 pyramidal neurons in the hippocampus. GABAergic postsynaptic currents (PSCs) were recorded in whole-cell voltage-clamped CA1 pyramidal neurons. By comparing spontaneous and miniature (action potential-independent) GABAergic PSCs, we found that multiple release site connectivity is established already at the first postnatal day and that the degree of connectivity remains unaltered into adulthood. During the same time there is a dramatic increase in the number of GABAergic synapses on each pyramidal neuron as indicated by the increase in frequency of miniature GABAergic PSCs. These results indicate that during development a given interneuron contacts an increasing number of target pyramidal neurons but with the same multiple release site connectivity. It has been shown previously that the connectivity between CA3 and CA1 pyramidal neurons is initially restricted to one release site, and develops gradually. The present result thus suggests different mechanisms to govern the maturation of excitatory and inhibitory synaptic transmissions.

  17. CosmoQuest Year 1.5: Citizen Scientist Behaviors and Site Usage Across Multiple Projects

    NASA Astrophysics Data System (ADS)

    Gugliucci, Nicole E.; Gay, P. L.; Bracey, G.; CosmoQuest Team

    2013-06-01

    CosmoQuest launched as a citizen science portal in January 2012 and has since expanded to include three projects in planetary surface mapping, one completed project searching for KBOs, and several more on the way with various astrophysical science goals. We take a close look at how our users move through the site, how much time they spend on various tasks, project retention rate, and how many use multiple projects on the site. We are also piloting a citizen science motivation survey given to random site users to find out why citizen scientists join new projects and continue to participate. This is part of a larger project using online and real-life interactions to study citizen scientist behaviors, motivations, and learning with a goal of building better community with researchers, volunteers, educators, and developers.

  18. Comparison of body composition assessment by hydrodensitometry, skinfolds, and multiple site near-infrared spectrophotometry.

    PubMed

    Hortobágyi, T; Israel, R G; Houmard, J A; McCammon, M R; O'Brien, K F

    1992-03-01

    Percent body fat (%BF) was assessed in 171 men with underwater weighing (UWW), seven-site skinfolds (7 SF), and near-infrared spectrophotometry (NIR). NIR was determined at eight sites, including biceps, triceps, axilla, chest, abdomen, suprailium, subscapula and thigh. Analysis of variance and Dunnett's post-hoc procedure revealed that NIR significantly (P less than 0.05) underpredicted %BF when using the biceps (12.9%), chest (11.3%), abdomen (10.2%), subscapula (11.3%) and thigh (9.9%) sites compared to the criterion %BF measured with UWW (13.4% +/- SD = 6.49). %BF with 7 SF was only 0.3% higher than %BF with UWW (r = 0.94, standard error of estimate = 2.9%). Correlation coefficients between SF thickness and NIR optical density readings at 940 nm (OD1) and 950 nm (OD2) wavelengths ranged from r = -0.30 (subscapula) to r = -0.67 (biceps) for OD1 and r = -0.39 (axilla) to r = -0.68 (biceps) for OD2. Multiple linear regression showed that the OD readings were not significant predictors of UWW %BF when using body mass, stature, activity level, and frame size as predictors, except for the OD2 reading at the biceps site. Stepwise regression revealed that NIR OD readings did not predict additional variance in %BF beyond mass, stature, activity level, frame size, or 7 SF. It was concluded that 7 SF are better predictors of %BF than NIR, and that NIR at multiple sites is inconsistent in assessing body composition.

  19. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  20. Multiple sites of methylation in the methyl accepting chemotaxis proteins of Escherichia coli

    SciTech Connect

    Chelsky, D.; Dahlquist, F.W.

    1981-01-01

    The methyl-accepting chemotaxis proteins (MCP) of E coli show at least five bands when subjected to SDS-gel electrophoresis. The intensity of the individual bands varies depending on the environment of the cells before solubilization. The faster migrating bands are enhanced following attractant stimulation, whereas the slower migrating bands are enhanced following attractant dilution or repellent increase. The time scale of these intensity changes is similar to that for adaptation of the behavioral response in these cells suggesting that methylation of the MCP is involved in producing these bands. Peptide mapping experiments show three methylated peptides in both MCP I and MCP II. These results suggest multiple sites of methylation, which are responsible, at least in part, for the observed multiple bands of the MCPs.

  1. Molecular characterization of Neisseria gonorrhoeae on non-cultured specimens from multiple anatomic sites.

    PubMed

    Carannante, Anna; Ghisetti, Valeria; Dal Conte, Ivano; Gregori, Gabriella; Stella, Maria Laura; Vacca, Paola; Del Re, Simonetta; Stefanelli, Paola

    2017-01-01

    The aim of this study was to molecularly characterize Neisseria gonorrhoeae on non-cultured specimens collected from multiple anatomic sites. N. gonorrhoeae multiantigen sequence typing (NG-MAST) together with the gene sequence analysis of antimicrobial resistance (AMR) target genes were used. Seventeen genital and extra-genital samples from eight patients (7 were men who have sex with men, MSM, and 1 women who have sex with men, WSM) with gonorrhoea symptoms were analyzed. For 7, of the 8 patients, conventional culture method has been used to identify gonorrhoea. All the samples were tested with the rapid molecular method CEPHEID. Amplification and sequencing of porB and tbpB, to identify the Sequence Type (ST) by NG-MAST, and penA, mtrR, porB1b, ponA genes were also performed. Antimicrobial susceptibility by Etest, for the available culture positive samples, was carried out. For 7 patients the ST was obtained and for 6 the complete sequence analysis of the AMR target genes was also defined. For the majority of them, samples collected from multiple sites (oropharynx, rectum, vaginal and urethra) confirm the presence of the same gonorrhoea strain. In particular, for 5 patients the same STs and changes in the AMR target genes were identified. Molecular characterization on non-cultured or culture negative specimens for gonorrhoea can successfully be applied directly to genital and extra-genital samples. Thus permit to identify the presence of the same strain in patients with gonorrhoea infection in multiple anatomic sites and to predict the antimicrobial susceptibility pattern.

  2. Multiple site receptor modeling with a minimal spanning tree combined with a Kohonen neural network

    NASA Astrophysics Data System (ADS)

    Hopke, Philip K.

    1999-12-01

    A combination of two pattern recognition methods has been developed that allows the generation of geographical emission maps form multivariate environmental data. In such a projection into a visually interpretable subspace by a Kohonen Self-Organizing Feature Map, the topology of the higher dimensional variables space can be preserved, but parts of the information about the correct neighborhood among the sample vectors will be lost. This can partly be compensated for by an additional projection of Prim's Minimal Spanning Tree into the trained neural network. This new environmental receptor modeling technique has been adapted for multiple sampling sites. The behavior of the method has been studied using simulated data. Subsequently, the method has been applied to mapping data sets from the Southern California Air Quality Study. The projection of a 17 chemical variables measured at up to 8 sampling sites provided a 2D, visually interpretable, geometrically reasonable arrangement of air pollution source sin the South Coast Air Basin.

  3. Hedgehog Pathway Modulation by Multiple Lipid Binding Sites on the Smoothened Effector of Signal Response

    PubMed Central

    Myers, Benjamin R.; Sever, Navdar; Chong, Yong Chun; Kim, James; Belani, Jitendra D.; Rychnovsky, Scott; Bazan, J. Fernando; Beachy, Philip A.

    2014-01-01

    Summary Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo), by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs. PMID:23954590

  4. Current DOT research on the effect of multiple site damage on structural integrity

    NASA Astrophysics Data System (ADS)

    Tong, P.; Arin, Kemal; Jeong, David Y.; Greif, R.; Brewer, John C.; Bobo, Stephan N.; Sampath, Sam N.

    1992-07-01

    Multiple site damage (MSD) is a type of cracking that may be found in aging airplanes and which may adversely affect their continuing airworthiness. The Volpe National Transportation Systems Center has supported the Federal Aviation Administration Technical Center on structural integrity research for the past two and half years. The work has focused on understanding the behavior of MSD, detection of MSD during airframe inspection, and the avoidance of MSD in future designs. These three elements of the MSD problem are addressed and a summary of the completed work, the current status, and requirements for future research is provided.

  5. Isolated Gastrointestinal Sarcoidosis Involving Multiple Gastrointestinal Sites Presenting as Chronic Diarrhea

    PubMed Central

    Gaye, Bilkisu; Makary, Raafat; Monteiro, Carmela; Eid, Emely

    2016-01-01

    Sarcoidosis is a chronic and systemic disorder characterized by the formation of non-caseating granulomas. Very few cases of isolated gastrointestinal sarcoidosis have been reported, and even fewer, if any, report gastrointestinal sarcoidosis within multiple gastrointestinal sites concomitantly. We present a 42-year-old white man with chronic diarrhea and abdominal pain for more than 3 years. Mucosal biopsies revealed non-caseating microgranulomas in the stomach, throughout the small intestine, colon, and rectum. Prednisone therapy was initiated with a rapid improvement in symptoms and complete resolution of diarrhea within 3 weeks. PMID:28119949

  6. Seeing the forest through the trees: Considering roost-site selection at multiple spatial scales

    USGS Publications Warehouse

    Jachowski, David S.; Rota, Christopher T.; Dobony, Christopher A.; Ford, W. Mark; Edwards, John W.

    2016-01-01

    Conservation of bat species is one of the most daunting wildlife conservation challenges in North America, requiring detailed knowledge about their ecology to guide conservation efforts. Outside of the hibernating season, bats in temperate forest environments spend their diurnal time in day-roosts. In addition to simple shelter, summer roost availability is as critical as maternity sites and maintaining social group contact. To date, a major focus of bat conservation has concentrated on conserving individual roost sites, with comparatively less focus on the role that broader habitat conditions contribute towards roost-site selection. We evaluated roost-site selection by a northern population of federally-endangered Indiana bats (Myotis sodalis) at Fort Drum Military Installation in New York, USA at three different spatial scales: landscape, forest stand, and individual tree level. During 2007–2011, we radiotracked 33 Indiana bats (10 males, 23 females) and located 348 roosting events in 116 unique roost trees. At the landscape scale, bat roost-site selection was positively associated with northern mixed forest, increased slope, and greater distance from human development. At the stand scale, we observed subtle differences in roost site selection based on sex and season, but roost selection was generally positively associated with larger stands with a higher basal area, larger tree diameter, and a greater sugar maple (Acer saccharum) component. We observed no distinct trends of roosts being near high-quality foraging areas of water and forest edges. At the tree scale, roosts were typically in American elm (Ulmus americana) or sugar maple of large diameter (>30 cm) of moderate decay with loose bark. Collectively, our results highlight the importance of considering day roost needs simultaneously across multiple spatial scales. Size and decay class of individual roosts are key ecological attributes for the Indiana bat, however, larger-scale stand structural

  7. Seeing the Forest through the Trees: Considering Roost-Site Selection at Multiple Spatial Scales.

    PubMed

    Jachowski, David S; Rota, Christopher T; Dobony, Christopher A; Ford, W Mark; Edwards, John W

    2016-01-01

    Conservation of bat species is one of the most daunting wildlife conservation challenges in North America, requiring detailed knowledge about their ecology to guide conservation efforts. Outside of the hibernating season, bats in temperate forest environments spend their diurnal time in day-roosts. In addition to simple shelter, summer roost availability is as critical as maternity sites and maintaining social group contact. To date, a major focus of bat conservation has concentrated on conserving individual roost sites, with comparatively less focus on the role that broader habitat conditions contribute towards roost-site selection. We evaluated roost-site selection by a northern population of federally-endangered Indiana bats (Myotis sodalis) at Fort Drum Military Installation in New York, USA at three different spatial scales: landscape, forest stand, and individual tree level. During 2007-2011, we radiotracked 33 Indiana bats (10 males, 23 females) and located 348 roosting events in 116 unique roost trees. At the landscape scale, bat roost-site selection was positively associated with northern mixed forest, increased slope, and greater distance from human development. At the stand scale, we observed subtle differences in roost site selection based on sex and season, but roost selection was generally positively associated with larger stands with a higher basal area, larger tree diameter, and a greater sugar maple (Acer saccharum) component. We observed no distinct trends of roosts being near high-quality foraging areas of water and forest edges. At the tree scale, roosts were typically in American elm (Ulmus americana) or sugar maple of large diameter (>30 cm) of moderate decay with loose bark. Collectively, our results highlight the importance of considering day roost needs simultaneously across multiple spatial scales. Size and decay class of individual roosts are key ecological attributes for the Indiana bat, however, larger-scale stand structural components

  8. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  9. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    NASA Astrophysics Data System (ADS)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-07-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

  10. Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

    PubMed

    Iturriaga-Vásquez, Patricio; Alzate-Morales, Jans; Bermudez, Isabel; Varas, Rodrigo; Reyes-Parada, Miguel

    2015-11-01

    For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.

  11. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: evidence for multiple estrogen binding sites

    SciTech Connect

    Lesch, K.P.; Schott, W.; Gross, S.

    1987-09-01

    The existence of estrogen receptors in human meningiomas has long been a controversial issue. This may be explained, in part, by apparent heterogeneity of estrogen binding sites in meningioma tissue. In this study, estrogen receptors were determined in 58 meningiomas with an enzyme immunoassay using monoclonal antibodies against human estrogen receptor protein (estrophilin) and with a sensitive radioligand binding assay using /sup 125/I-labeled estradiol (/sup 125/I-estradiol) as radioligand. Low levels of estrophilin immunoreactivity were found in tumors from 62% of patients, whereas radioligand binding activity was demonstrated in about 46% of the meningiomas examined. In eight (14%) tissue samples multiple binding sites for estradiol were observed. The immunoreactive binding sites correspond to the classical, high affinity estrogen receptors: the Kd for /sup 125/I-estradiol binding to the receptor was approximately 0.2 nM and the binding was specific for estrogens. The second, low affinity class of binding sites considerably influenced measurement of the classical receptor even at low ligand concentrations. The epidemiological and clinical data from patients with meningiomas, and the existence of specific estrogen receptors confirmed by immunochemical detection, may be important factors in a theory of oncogenesis.

  12. Source apportionment of PM2.5 size distribution and composition data from multiple stationary sites using a mobile platform

    NASA Astrophysics Data System (ADS)

    Liao, Ho-Tang; Chou, Charles C.-K.; Huang, Sheng-Hsiu; Lu, Chia-Jung; Chen, Chih-Chieh; Hopke, Philip K.; Wu, Chang-Fu

    2017-07-01

    Several source apportionment studies considering multiple sites showed spatial variability of source contributions. However, setting up multiple fixed sites to collect comprehensive chemical speciation data is resource demanding. In this study, field campaigns were conducted at multiple receptor sites in the Mailiao and Taishi townships in Yunlin County using a mobile platform to demonstrate the feasibility of receptor modeling with particle size distribution and PM2.5 speciation data. Sources of air pollutants to all of the monitoring sites were identified and quantified using a modified positive matrix factorization (PMF) model. Modeling results indicated that a mixed source dominated by secondary aerosol was the largest contributor to PM2.5 at most sites. Adding VOC measurements with high time resolution helped to improve the source separation. Different patterns of source contributions among sites and seasons were observed showing both spatial heterogeneity and seasonal variation.

  13. Amyloid tracers detect multiple binding sites in Alzheimer's disease brain tissue.

    PubMed

    Ni, Ruiqing; Gillberg, Per-Göran; Bergfors, Assar; Marutle, Amelia; Nordberg, Agneta

    2013-07-01

    -1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

  14. Multiple site-selective insertions of non-canonical amino acids into sequence-repetitive polypeptides

    PubMed Central

    Wu, I-Lin; Patterson, Melissa A.; Carpenter Desai, Holly E.; Mehl, Ryan A.; Giorgi, Gianluca

    2013-01-01

    A simple and efficient method is described for introduction of non-canonical amino acids at multiple, structurally defined sites within recombinant polypeptide sequences. E. coli MRA30, a bacterial host strain with attenuated activity for release factor 1 (RF1), is assessed for its ability to support the incorporation of a diverse range of non-canonical amino acids in response to multiple encoded amber (TAG) codons within genetic templates derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and isolated protein yield were observed to depend on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNACUA pair and the non-canonical amino acid substrate. This approach afforded elastin-mimetic protein polymers containing non-canonical amino acid derivatives at up to twenty-two positions within the repeat sequence with high levels of substitution. The identity and position of the variant residues was confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments resulting from thermolysin digestion. The accumulated data suggest that this multi-site suppression approach permits the preparation of protein-based materials in which novel chemical functionality can be introduced at precisely defined positions within the polypeptide sequence. PMID:23625817

  15. Microbiome Assembly across Multiple Body Sites in Low-Birthweight Infants

    PubMed Central

    Costello, Elizabeth K.; Carlisle, Erica M.; Bik, Elisabeth M.; Morowitz, Michael J.; Relman, David A.

    2013-01-01

    ABSTRACT The purpose of this study was to evaluate the composition and richness of bacterial communities associated with low-birthweight (LBW) infants in relation to host body site, individual, and age. Bacterial 16S rRNA genes from saliva samples, skin swabs, and stool samples collected on postnatal days 8, 10, 12, 15, 18, and 21 from six LBW (five premature) infants were amplified, pyrosequenced, and analyzed within a comparative framework that included analogous data from normal-birthweight (NBW) infants and healthy adults. We found that body site was the primary determinant of bacterial community composition in the LBW infants. However, site specificity depended on postnatal age: saliva and stool compositions diverged over time but were not significantly different until the babies were 15 days old. This divergence was primarily driven by progressive temporal turnover in the distal gut, which proceeded at a rate similar to that of age-matched NBW infants. Neonatal skin was the most adult-like in microbiota composition, while saliva and stool remained the least so. Compositional variation among infants was marked and depended on body site and age. Only the smallest, most premature infant received antibiotics during the study period; this heralded a coexpansion of Pseudomonas aeruginosa and a novel Mycoplasma sp. in the oral cavity of this vaginally delivered, intubated patient. We conclude that concurrent molecular surveillance of multiple body sites in LBW neonates reveals a delayed compositional differentiation of the oral cavity and distal gut microbiota and, in the case of one infant, an abundant, uncultivated oral Mycoplasma sp., recently detected in human vaginal samples. PMID:24169577

  16. Multiple ion binding sites in Ih channels of rod photoreceptors from tiger salamanders.

    PubMed

    Wollmuth, L P

    1995-05-01

    The mechanism of ion permeation in K+/Na(+)-permeable Ih channels of tiger salamander rod photoreceptors was investigated using the whole-cell voltage-clamp technique. Ih channels showed features indicative of pores with multiple ion binding sites: in mixtures of K+ and thallium (T1+), the amplitude of the time-dependent current showed an anomalous mole fraction dependence, and K+ permeation was blocked by other permeant ions (with K0.5 values: T1+, 44 microM; Rb+, 220 microM and NH4+, 1100 microM) as well as by essentially impermeant ions (Cs+, 22 microM Ba2+, 9200 microM) which apparently block Ih by binding in the pore. In contrast, Na+ had little blocking action on K+ permeation. The block by all of these ions was sensitive to external K+ with the block by Cs+ being the least sensitive. Na+ was more effective than K+ in reducing the block by T1+, Rb+ and NH4+, but was less effective for the block by Cs+ and Ba2+. The blocking action of Cs+ and Ba2+ was non-competitive, suggesting that they block Ih channels at independent sites. Based on the efficacy of block by the different ions, the degree to which K+ and Na+ antagonize this block and the noncompetitive blocking action of Cs+ and Ba2+, the permeation pathway of Ih channels appears to contain at least three ion binding sites with at least two sites having a higher affinity for K+ over Na+ and another site with a higher affinity for Na+ over K+.

  17. Prevention of adolescent reoccurring violence and alcohol abuse: a multiple site evaluation.

    PubMed

    Wodarski, John S

    2010-07-01

    "Prevention of Adolescent Reoccurring Violence and Alcohol Abuse: A Multiple Site Evaluation" is a multiple component alcohol abuse and violent behavior prevention strategy, targeted to adolescents ages 16-21 who have high levels of anger, or who are victims/perpetrators of violence, and their families. Three community centers located in upstate New York provided group participants (N = 210) known to have conduct disorder and substance abuse history. The centers were used as the intervention sites over a seven-week period with the youth assessment staff using objective screening measures. The participants were exposed to a two-pronged intervention, using a parental involvement cohort with approximately half of the study participants. The Teams, Games, and Tournaments strategy was the intervention method. Teams, Games, and Tournaments is a Social Learning Theory-based intervention with demonstrated empirical evidence of the model's effectiveness. A 2 x 3 factorial design with two follow-up points encompassed: anger control, alcohol/substance abuse, and family interactive education. The goals of the study were to help adolescents reduce their alcohol use, to increase productive family interaction, and ultimately to reduce the adolescents' aggression levels and subsequently reduce the possibility of their becoming victims or perpetrators of a violent crime. Consistent with Social Learning Theory, the Teams, Games, and Tournaments treatment intervention makes use of adolescents as peer counselors. The practical implications include that professionals or students in our public schools, juvenile courts, correctional institutions, and residential treatment centers can easily implement this program. A standardized treatment manual is available. It offers a complete, ready-to-use, and cost-effective tool for reducing adolescent violence and alcohol abuse. Further, the data provide support for a hypothesis of social learning theory, that is: interventions using multiple

  18. GFI1B controls its own expression binding to multiple sites

    PubMed Central

    Anguita, Eduardo; Villegas, Ana; Iborra, Francisco; Hernández, Aurora

    2010-01-01

    Background Transcription factors play essential roles in both normal and malignant hematopoiesis. This is the case for the growth factor independent 1b (GFI1B) transcription factor, which is required for erythroid and megakaryocytic differentiation and over-expressed in leukemic patients and cell lines. Design and Methods To investigate GFI1B regulation, we searched for multispecies conserved non-coding elements between GFI1B and neighboring genes. We used a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay and DNase1 hypersensitivity to assess the chromatin conformation of these sites. Next, we analyzed transcription factor binding and histone modifications at the GFI1B locus including the conserved non-coding elements by a chromatin immunoprecipitation assay. Finally, we studied the interaction of the GFI1B promoter and the conserved non-coding elements with the chromatin conformation capture technique and used immunofluorescence to evaluate GFI1B levels in individual cells. Results We localized several conserved non-coding elements containing multiple erythroid specific transcription factor binding sites at the GFI1B locus. In GFI1B-expressing cells a subset of these conserved non-coding elements and the promoter adopt a close spatial conformation, localize with open chromatin sites, harbor chromatin modifications associated with gene activation and bind multiple transcription factors and co-repressors. Conclusions Our findings indicate that GFI1B regulatory elements behave as activators and repressors. Different protein levels within a cell population suggest that cells must activate and repress GFI1B continuously to control its final level. These data are consistent with a model of GFI1B regulation in which GFI1B binds to its own promoter and to the conserved non-coding elements as its levels rise. This would attract repressor complexes that progressively down-regulate the gene. GFI1B expression would decrease until a stage at which the

  19. GFI1B controls its own expression binding to multiple sites.

    PubMed

    Anguita, Eduardo; Villegas, Ana; Iborra, Francisco; Hernández, Aurora

    2010-01-01

    Transcription factors play essential roles in both normal and malignant hematopoiesis. This is the case for the growth factor independent 1b (GFI1B) transcription factor, which is required for erythroid and megakaryocytic differentiation and over-expressed in leukemic patients and cell lines. To investigate GFI1B regulation, we searched for multispecies conserved non-coding elements between GFI1B and neighboring genes. We used a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay and DNase1 hypersensitivity to assess the chromatin conformation of these sites. Next, we analyzed transcription factor binding and histone modifications at the GFI1B locus including the conserved non-coding elements by a chromatin immunoprecipitation assay. Finally, we studied the interaction of the GFI1B promoter and the conserved non-coding elements with the chromatin conformation capture technique and used immunofluorescence to evaluate GFI1B levels in individual cells. We localized several conserved non-coding elements containing multiple erythroid specific transcription factor binding sites at the GFI1B locus. In GFI1B-expressing cells a subset of these conserved non-coding elements and the promoter adopt a close spatial conformation, localize with open chromatin sites, harbor chromatin modifications associated with gene activation and bind multiple transcription factors and co-repressors. Conclusions Our findings indicate that GFI1B regulatory elements behave as activators and repressors. Different protein levels within a cell population suggest that cells must activate and repress GFI1B continuously to control its final level. These data are consistent with a model of GFI1B regulation in which GFI1B binds to its own promoter and to the conserved non-coding elements as its levels rise. This would attract repressor complexes that progressively down-regulate the gene. GFI1B expression would decrease until a stage at which the activating complexes predominate and

  20. Chloroplast group III twintron excision utilizing multiple 5'- and 3'-splice sites.

    PubMed Central

    Copertino, D W; Shigeoka, S; Hallick, R B

    1992-01-01

    The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed. Images PMID:1464326

  1. Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.

    PubMed

    Thompson, Karol L; Boitier, Eric; Chen, Tao; Couttet, Philippe; Ellinger-Ziegelbauer, Heidrun; Goetschy, Manuela; Guillemain, Gregory; Kanki, Masayuki; Kelsall, Janet; Mariet, Claire; de La Moureyre-Spire, Catherine; Mouritzen, Peter; Nassirpour, Rounak; O'Lone, Raegan; Pine, P Scott; Rosenzweig, Barry A; Sharapova, Tatiana; Smith, Aaron; Uchiyama, Hidefumi; Yan, Jian; Yuen, Peter S; Wolfinger, Russ

    2016-11-01

    Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs. Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.

  2. Disruption of the microbiota across multiple body sites in critically ill children.

    PubMed

    Rogers, Matthew B; Firek, Brian; Shi, Min; Yeh, Andrew; Brower-Sinning, Rachel; Aveson, Victoria; Kohl, Brittany L; Fabio, Anthony; Carcillo, Joseph A; Morowitz, Michael J

    2016-12-29

    Despite intense interest in the links between the microbiome and human health, little has been written about dysbiosis among ICU patients. We characterized microbial diversity in samples from 37 children in a pediatric ICU (PICU). Standard measures of alpha and beta diversity were calculated, and results were compared with data from adult and pediatric reference datasets. Bacterial 16S rRNA gene sequences were analyzed from 71 total tongue swabs, 50 skin swabs, and 77 stool samples or rectal swabs. The mean age of the PICU patients was 2.9 years (range 1-9 years), and many were chronically ill children that had previously been hospitalized in the PICU. Relative to healthy adults and children, alpha diversity was decreased in PICU GI and tongue but not skin samples. Measures of beta diversity indicated differences in community membership at each body site between PICU, adult, and pediatric groups. Taxonomic alterations in the PICU included enrichment of gut pathogens such as Enterococcus and Staphylococcus at multiple body sites and depletion of commensals such as Faecalibacterium and Ruminococcus from GI samples. Alpha and beta diversity were unstable over time in patients followed longitudinally. We observed the frequent presence of "dominant" pathogens in PICU samples at relative abundance >50%. PICU samples were characterized by loss of site specificity, with individual taxa commonly present simultaneously at three sample sites on a single individual. Some pathogens identified by culture of tracheal aspirates were commonly observed in skin samples from the same patient. We conclude that the microbiota in critically ill children differs sharply from the microbiota of healthy children and adults. Acknowledgement of dysbiosis associated with critical illness could provide opportunities to modulate the microbiota with precision and thereby improve patient outcomes.

  3. Seeing the Forest through the Trees: Considering Roost-Site Selection at Multiple Spatial Scales

    PubMed Central

    Jachowski, David S.; Rota, Christopher T.; Dobony, Christopher A.; Ford, W. Mark; Edwards, John W.

    2016-01-01

    Conservation of bat species is one of the most daunting wildlife conservation challenges in North America, requiring detailed knowledge about their ecology to guide conservation efforts. Outside of the hibernating season, bats in temperate forest environments spend their diurnal time in day-roosts. In addition to simple shelter, summer roost availability is as critical as maternity sites and maintaining social group contact. To date, a major focus of bat conservation has concentrated on conserving individual roost sites, with comparatively less focus on the role that broader habitat conditions contribute towards roost-site selection. We evaluated roost-site selection by a northern population of federally-endangered Indiana bats (Myotis sodalis) at Fort Drum Military Installation in New York, USA at three different spatial scales: landscape, forest stand, and individual tree level. During 2007–2011, we radiotracked 33 Indiana bats (10 males, 23 females) and located 348 roosting events in 116 unique roost trees. At the landscape scale, bat roost-site selection was positively associated with northern mixed forest, increased slope, and greater distance from human development. At the stand scale, we observed subtle differences in roost site selection based on sex and season, but roost selection was generally positively associated with larger stands with a higher basal area, larger tree diameter, and a greater sugar maple (Acer saccharum) component. We observed no distinct trends of roosts being near high-quality foraging areas of water and forest edges. At the tree scale, roosts were typically in American elm (Ulmus americana) or sugar maple of large diameter (>30 cm) of moderate decay with loose bark. Collectively, our results highlight the importance of considering day roost needs simultaneously across multiple spatial scales. Size and decay class of individual roosts are key ecological attributes for the Indiana bat, however, larger-scale stand structural

  4. Uncertainty analysis of the Operational Simplified Surface Energy Balance (SSEBop) model at multiple flux tower sites

    NASA Astrophysics Data System (ADS)

    Chen, Mingshi; Senay, Gabriel B.; Singh, Ramesh K.; Verdin, James P.

    2016-05-01

    Evapotranspiration (ET) is an important component of the water cycle - ET from the land surface returns approximately 60% of the global precipitation back to the atmosphere. ET also plays an important role in energy transport among the biosphere, atmosphere, and hydrosphere. Current regional to global and daily to annual ET estimation relies mainly on surface energy balance (SEB) ET models or statistical and empirical methods driven by remote sensing data and various climatological databases. These models have uncertainties due to inevitable input errors, poorly defined parameters, and inadequate model structures. The eddy covariance measurements on water, energy, and carbon fluxes at the AmeriFlux tower sites provide an opportunity to assess the ET modeling uncertainties. In this study, we focused on uncertainty analysis of the Operational Simplified Surface Energy Balance (SSEBop) model for ET estimation at multiple AmeriFlux tower sites with diverse land cover characteristics and climatic conditions. The 8-day composite 1-km MODerate resolution Imaging Spectroradiometer (MODIS) land surface temperature (LST) was used as input land surface temperature for the SSEBop algorithms. The other input data were taken from the AmeriFlux database. Results of statistical analysis indicated that the SSEBop model performed well in estimating ET with an R2 of 0.86 between estimated ET and eddy covariance measurements at 42 AmeriFlux tower sites during 2001-2007. It was encouraging to see that the best performance was observed for croplands, where R2 was 0.92 with a root mean square error of 13 mm/month. The uncertainties or random errors from input variables and parameters of the SSEBop model led to monthly ET estimates with relative errors less than 20% across multiple flux tower sites distributed across different biomes. This uncertainty of the SSEBop model lies within the error range of other SEB models, suggesting systematic error or bias of the SSEBop model is within the

  5. Uncertainty analysis of the Operational Simplified Surface Energy Balance (SSEBop) model at multiple flux tower sites

    USGS Publications Warehouse

    Chen, Mingshi; Senay, Gabriel B.; Singh, Ramesh K.; Verdin, James P.

    2016-01-01

    Evapotranspiration (ET) is an important component of the water cycle – ET from the land surface returns approximately 60% of the global precipitation back to the atmosphere. ET also plays an important role in energy transport among the biosphere, atmosphere, and hydrosphere. Current regional to global and daily to annual ET estimation relies mainly on surface energy balance (SEB) ET models or statistical and empirical methods driven by remote sensing data and various climatological databases. These models have uncertainties due to inevitable input errors, poorly defined parameters, and inadequate model structures. The eddy covariance measurements on water, energy, and carbon fluxes at the AmeriFlux tower sites provide an opportunity to assess the ET modeling uncertainties. In this study, we focused on uncertainty analysis of the Operational Simplified Surface Energy Balance (SSEBop) model for ET estimation at multiple AmeriFlux tower sites with diverse land cover characteristics and climatic conditions. The 8-day composite 1-km MODerate resolution Imaging Spectroradiometer (MODIS) land surface temperature (LST) was used as input land surface temperature for the SSEBop algorithms. The other input data were taken from the AmeriFlux database. Results of statistical analysis indicated that the SSEBop model performed well in estimating ET with an R2 of 0.86 between estimated ET and eddy covariance measurements at 42 AmeriFlux tower sites during 2001–2007. It was encouraging to see that the best performance was observed for croplands, where R2 was 0.92 with a root mean square error of 13 mm/month. The uncertainties or random errors from input variables and parameters of the SSEBop model led to monthly ET estimates with relative errors less than 20% across multiple flux tower sites distributed across different biomes. This uncertainty of the SSEBop model lies within the error range of other SEB models, suggesting systematic error or bias of the SSEBop model is within

  6. A density functional theory for colloids with two multiple bonding associating sites.

    PubMed

    Haghmoradi, Amin; Wang, Le; Chapman, Walter G

    2016-06-22

    Wertheim's multi-density formalism is extended for patchy colloidal fluids with two multiple bonding patches. The theory is developed as a density functional theory to predict the properties of an associating inhomogeneous fluid. The equation of state developed for this fluid depends on the size of the patch, and includes formation of cyclic, branched and linear clusters of associated species. The theory predicts the density profile and the fractions of colloids in different bonding states versus the distance from one wall as a function of bulk density and temperature. The predictions from our theory are compared with previous results for a confined fluid with four single bonding association sites. Also, comparison between the present theory and Monte Carlo simulation indicates a good agreement.

  7. A Novel Method for Simulating Stochastic Simulations of Daily Precipitation and Air Temperature at Multiple Sites

    NASA Astrophysics Data System (ADS)

    Smith, K.; Strong, C.; Rassoul-Agha, F.

    2016-12-01

    A parametric stochastic weather generator (SWG) is introduced that simulates trended, nonstationary precipitation and temperature values directly, circumventing the conventional approach of adding simulated standardized anomalies of temperature to a prescribed cyclostationary mean. The model mean makes autocorrelated transitions between wet- and dry-state values, and its parameters are determined by optimizing harmonic and trend terms. If the stochastic ("noise") term is assumed to have constant amplitude, analytical results are available via maximum likelihood estimation (MLE) and are equivalent to least squares estimation (LSE). Where observations motivate a seasonally-varying noise coefficient, MLE becomes nonlinear, and we formulate an analytical solution via LSE. For illustration, the SWG is shown to produce realistic representations of daily precipitation and maximum and minimum air temperature at multiple sites, which for the study includes the eastern Great Basin in Northern Utah.

  8. A laboratory study of fracture in the presence of lap splice multiple site damage

    NASA Astrophysics Data System (ADS)

    Mayville, Ronald A.; Warren, Thomas J.

    Flat coupons were tested in the laboratory to determine a fracture criterion for link-up of fuselage lap splice multiple site damage at adjacent rivet holes. Experiments were performed on 0.040 inch (1 mm) thick 2024-T3 clad aluminum sheet. Continuous and riveted lap splice coupons were tested with simulated uniform (equal crack lengths) and nonuniform MSD, and the effects of notch sharpness were also studied. A net section stress criterion was found to provide excellent predictions of fracture for uniform MSD and uniform stress distributions. This same criterion provides conservative predictions for nonuniform MSD in uniform stress fields. An overload/cyclic stress experiment was also conducted to explore the pressurized proof test scenario of ensuring structural integrity.

  9. CpG island hypermethylation at multiple gene sites in diagnosis and prognosis of prostate cancer.

    PubMed

    Ellinger, Jörg; Bastian, Patrick J; Jurgan, Thomas; Biermann, Katharina; Kahl, Philip; Heukamp, Lukas C; Wernert, Nicolas; Müller, Stefan C; von Ruecker, Alexander

    2008-01-01

    CpG island hypermethylation causes gene silencing and could be decisive in prostate carcinogenesis and progression. We investigated its role at multiple gene sites during prostate carcinogenesis. A quantitative, methylation-specific polymerase chain reaction was used to analyze the hypermethylation patterns at nine gene loci (Annexin2, APC, EDNRB, GSTP1, PTGS2, MDR1, RARbeta, Reprimo, and TIG1) in 80 patients with prostate cancer (PCa) and 26 patients with benign prostatic hyperplasia (BPH). Hypermethylation was more frequent in PCa than in BPH tissues (EDNRB, 100% versus 88%; TIG1, 96% versus 12%; RARbeta, 95% versus 35%; GSTP1, 93% versus 15%; APC, 80% versus 50%; MDR1, 80% versus 31%; PTGS2, 68% versus 15%; Reprimo, 59% versus 19%; and Annexin2, 4% versus 0%). TIG1 and GSTP1 hypermethylation distinguished between PCa and BPH with a specificity of greater than 85% and sensitivity of greater than 93%. Hypermethylation at a single gene locus did not correlate with any clinicopathologic variables. In contrast, hypermethylation at two genes (eg, APC and TIG1, APC and GSTP1, APC and PTGS2, APC or MDR, GSTP1 or PTGS2) correlated significantly with the pathologic stage and/or Gleason score (P = 0.033 to 0.045). Hypermethylation at APC and Reprimo, as well as DNA hypermethylation at more than five genes, correlated significantly with the rate of prostate-specific antigen recurrence after radical prostatectomy (P = 0.0078 and P = 0.0074, respectively). Our results have confirmed that the hypermethylation patterns are helpful in the diagnosis and prognosis of PCa. Increases in CpG island hypermethylation at multiple gene sites occur during PCa progression and indicate early biochemical recurrence after radical prostatectomy.

  10. Multiple site-specific infrared dichroism of CD3-zeta, a transmembrane helix bundle.

    PubMed

    Torres, Jaume; Briggs, John A G; Arkin, Isaiah T

    2002-02-15

    The structure of the transmembrane domain of CD3-zeta a component of the T-cell receptor involved in signal transduction, has been studied in its native state (a lipid bilayer) by multiple site-specific infrared dichroism. For the first time, the transmembrane domain has been labelled at multiple positions along the sequence, representing a total of 11 samples, each labelled at a different residue with an isotopically modified carbonyl group, (13)C [double bond] (18)O. A strategy is outlined that, based on the above data, can yield the rotational orientation and the local helix tilt for each labelled residue, giving a detailed description of helix geometry. The results obtained indicate that the transmembrane segment is in an alpha-helical conformation throughout, with an average helix tilt of 12 degrees. The N-terminal side of the helix is more tilted than the C-terminal. In an accompanying paper we describe the implementation of the infrared data in a model-building study of the CD3-zeta transmembrane complex. The model obtained is entirely consistent with results based on evolutionary conservation data. Taken together, this study represents the first step towards elucidation of the backbone structure of a transmembrane alpha-helical bundle by infrared spectroscopy.

  11. Multiple heparan sulfate binding site engagements are required for the infectious entry of human papillomavirus type 16.

    PubMed

    Richards, Kathleen F; Bienkowska-Haba, Malgorzata; Dasgupta, Jhimli; Chen, Xiaojiang S; Sapp, Martin

    2013-11-01

    Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.

  12. Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16

    PubMed Central

    Richards, Kathleen F.; Bienkowska-Haba, Malgorzata; Dasgupta, Jhimli; Chen, Xiaojiang S.

    2013-01-01

    Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating. PMID:23966387

  13. Precision Construction of 2D Heteropore Covalent Organic Frameworks by a Multiple-Linking-Site Strategy.

    PubMed

    Qian, Cheng; Xu, Shun-Qi; Jiang, Guo-Fang; Zhan, Tian-Guang; Zhao, Xin

    2016-12-05

    Integrating different kinds of pores into one covalent organic framework (COF) endows it with hierarchical porosity and thus generates a member of a new class of COFs, namely, heteropore COFs. Whereas the construction of COFs with homoporosity has already been well developed, the fabrication of heteropore COFs still faces great challenges. Although two strategies have recently been developed to successfully construct heteropore COFs from noncyclic building blocks, they suffer from the generation of COF isomers, which decreases the predictability and controllability of construction of this type of reticular materials. In this work, this drawback was overcome by a multiple-linking-site strategy that offers precision construction of heteropore COFs containing two kinds of hexagonal pores with different shapes and sizes. This strategy was developed by designing a building block in which double linking sites are introduced at each branch of a C3 -symmetric skeleton, the most widely used scaffold to construct COFs with homogeneous porosity. This design provides a general way to precisely construct heteropore COFs without formation of isomers. Furthermore, the as-prepared heteropore COFs have hollow-spherical morphology, which has rarely been observed for COFs, and an uncommon staggered AB stacking was observed for the layers of the 2D heteropore COFs.

  14. Hyperactive antifreeze protein from fish contains multiple ice-binding sites.

    PubMed

    Graham, Laurie A; Marshall, Christopher B; Lin, Feng-Hsu; Campbell, Robert L; Davies, Peter L

    2008-02-19

    Antifreeze proteins (AFPs) are produced to prevent freezing in many fish species that are exposed to icy seawater. There are a number of nonhomologous types of AFPs, diverse in both sequence and structure, which share the function of binding to ice and inhibiting its growth. We recently discovered a hyperactive AFP in the winter flounder and related species that is many-fold more active than other fish AFPs. Like the 3-4-kDa type I AFPs, it is alanine-rich and highly helical, but this 17-kDa protein is considerably larger and forms a dimer. We have sequenced the cDNA encoding this new AFP to gain insight into its structure and evolutionary relationship to the type I AFP family. The gene is clearly homologous to the righteye flounder type I AFP genes. Thus we have designated this protein "hyperactive type I AFP" (hyp-type I). The sequence of hyp-type I AFP supports a structural model in which two extended 195-amino acid alpha-helices form an amphipathic homodimer with a series of linked Ala- and Thr-rich patches on the surface of the dimer, each of which resembles ice-binding sites of type I AFPs. The superior activity of hyp-type I AFP may derive from the large combined surface area of the ice-binding sites, recognition of multiple planes of ice, and protection of the basal plane from ice growth.

  15. Multiple-methods investigation of recharge at a humid-region fractured rock site, Pennsylvania, USA

    USGS Publications Warehouse

    Heppner, C.S.; Nimmo, J.R.; Folmar, G.J.; Gburek, W.J.; Risser, D.W.

    2007-01-01

    Lysimeter-percolate and well-hydrograph analyses were combined to evaluate recharge for the Masser Recharge Site (central Pennsylvania, USA). In humid regions, aquifer recharge through an unconfined low-porosity fractured-rock aquifer can cause large magnitude water-table fluctuations over short time scales. The unsaturated hydraulic characteristics of the subsurface porous media control the magnitude and timing of these fluctuations. Data from multiple sets of lysimeters at the site show a highly seasonal pattern of percolate and exhibit variability due to both installation factors and hydraulic property heterogeneity. Individual event analysis of well hydrograph data reveals the primary influences on water-table response, namely rainfall depth, rainfall intensity, and initial water-table depth. Spatial and seasonal variability in well response is also evident. A new approach for calculating recharge from continuous water-table elevation records using a master recession curve (MRC) is demonstrated. The recharge estimated by the MRC approach when assuming a constant specific yield is seasonal to a lesser degree than the recharge estimate resulting from the lysimeter analysis. Partial reconciliation of the two recharge estimates is achieved by considering a conceptual model of flow processes in the highly-heterogeneous underlying fractured porous medium. ?? Springer-Verlag 2007.

  16. Multiple well-shutdown tests and site-scale flow simulation in fractured rocks.

    PubMed

    Tiedeman, Claire R; Lacombe, Pierre J; Goode, Daniel J

    2010-01-01

    A new method was developed for conducting aquifer tests in fractured-rock flow systems that have a pump-and-treat (P&T) operation for containing and removing groundwater contaminants. The method involves temporary shutdown of individual pumps in wells of the P&T system. Conducting aquifer tests in this manner has several advantages, including (1) no additional contaminated water is withdrawn, and (2) hydraulic containment of contaminants remains largely intact because pumping continues at most wells. The well-shutdown test method was applied at the former Naval Air Warfare Center (NAWC), West Trenton, New Jersey, where a P&T operation is designed to contain and remove trichloroethene and its daughter products in the dipping fractured sedimentary rocks underlying the site. The detailed site-scale subsurface geologic stratigraphy, a three-dimensional MODFLOW model, and inverse methods in UCODE_2005 were used to analyze the shutdown tests. In the model, a deterministic method was used for representing the highly heterogeneous hydraulic conductivity distribution and simulations were conducted using an equivalent porous media method. This approach was very successful for simulating the shutdown tests, contrary to a common perception that flow in fractured rocks must be simulated using a stochastic or discrete fracture representation of heterogeneity. Use of inverse methods to simultaneously calibrate the model to the multiple shutdown tests was integral to the effectiveness of the approach.

  17. Multiple nodulation genes are up-regulated during establishment of reniform nematode feeding sites in soybean.

    PubMed

    Redding, Nathan Wayne; Agudelo, Paula; Wells, Christina E

    2017-09-15

    The semi-endoparastic reniform nematode (Rotylenchulus reniformis) infects over 300 plant species. Females penetrate host roots and induce formation of complex, multinucleate feeding sites called syncytia. While anatomical changes associated with reniform nematode infection are well documented, little is known about their molecular basis. We grew soybean (Glycine max) in a split-root growth system, inoculated half of each root system with R. reniformis, and quantified gene expression in infected and control root tissue at four dates after inoculation. Over 6,000 genes were differentially expressed between inoculated and control roots on at least one date (FDR = 0.01, |log2FC| ≥ 1), and 507 gene sets were significantly enriched or depleted in inoculated roots (FDR = 0.05). Numerous genes up-regulated during syncytium formation had previously been associated with rhizobia nodulation. These included the nodule-initiating transcription factors CYCLOPS, NSP1, NSP2, and NIN, as well as multiple nodulins associated with the plant-derived peribacteroid membrane. Nodulation-related NIP aquaporins and SWEET sugar transporters were induced, as were plant CLAVATA3/ESR-related (CLE) signaling proteins and cell cycle regulators such as CCS52A and E2F. Nodulins and nodule-associated genes may have ancestral functions in normal root development and mycorrhization that have been co-opted by both parasitic nematodes and rhizobial bacteria to promote feeding site and nodule formation.

  18. Multiple well-shutdown tests and site-scale flow simulation in fractured rocks

    USGS Publications Warehouse

    Tiedeman, Claire R.; Lacombe, Pierre J.; Goode, Daniel J.

    2010-01-01

    A new method was developed for conducting aquifer tests in fractured-rock flow systems that have a pump-and-treat (P&T) operation for containing and removing groundwater contaminants. The method involves temporary shutdown of individual pumps in wells of the P&T system. Conducting aquifer tests in this manner has several advantages, including (1) no additional contaminated water is withdrawn, and (2) hydraulic containment of contaminants remains largely intact because pumping continues at most wells. The well-shutdown test method was applied at the former Naval Air Warfare Center (NAWC), West Trenton, New Jersey, where a P&T operation is designed to contain and remove trichloroethene and its daughter products in the dipping fractured sedimentary rocks underlying the site. The detailed site-scale subsurface geologic stratigraphy, a three-dimensional MODFLOW model, and inverse methods in UCODE_2005 were used to analyze the shutdown tests. In the model, a deterministic method was used for representing the highly heterogeneous hydraulic conductivity distribution and simulations were conducted using an equivalent porous media method. This approach was very successful for simulating the shutdown tests, contrary to a common perception that flow in fractured rocks must be simulated using a stochastic or discrete fracture representation of heterogeneity. Use of inverse methods to simultaneously calibrate the model to the multiple shutdown tests was integral to the effectiveness of the approach.

  19. Force mapping during the formation and maturation of cell adhesion sites with multiple optical tweezers.

    PubMed

    Schwingel, Melanie; Bastmeyer, Martin

    2013-01-01

    Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.

  20. Force Mapping during the Formation and Maturation of Cell Adhesion Sites with Multiple Optical Tweezers

    PubMed Central

    Schwingel, Melanie; Bastmeyer, Martin

    2013-01-01

    Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity. PMID:23372781

  1. Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

    PubMed

    Kim, Su Jin; Joo, Jeong Chan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

    2015-04-01

    Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack. © 2014 Wiley Periodicals, Inc.

  2. Prevalence of avian haemosporidian parasites is positively related to the abundance of host species at multiple sites within a region.

    PubMed

    Ellis, Vincenzo A; Medeiros, Matthew C I; Collins, Michael D; Sari, Eloisa H R; Coffey, Elyse D; Dickerson, Rebecca C; Lugarini, Camile; Stratford, Jeffrey A; Henry, Donata R; Merrill, Loren; Matthews, Alix E; Hanson, Alison A; Roberts, Jackson R; Joyce, Michael; Kunkel, Melanie R; Ricklefs, Robert E

    2017-01-01

    Parasite prevalence is thought to be positively related to host population density owing to enhanced contagion. However, the relationship between prevalence and local abundance of multiple host species is underexplored. We surveyed birds and their haemosporidian parasites (genera Plasmodium and Haemoproteus) at multiple sites across eastern North America to test whether the prevalence of these parasites in a host species at a particular site is related to that host's local abundance. Prevalence was positively related to host abundance within most sites, although the effect was stronger and more consistent for Plasmodium than for Haemoproteus. In contrast, prevalence was not related to variation in the abundance of most individual host species among sites across the region. These results suggest that parasite prevalence partly reflects the relative abundances of host species in local assemblages. However, three nonnative host species had low prevalence despite being relatively abundant at one site, as predicted by the enemy release hypothesis.

  3. Human Papillomavirus at Multiple Sites Associated with Anal Squamous Intraepithelial Lesions in HIV-Seropositive Individuals

    PubMed Central

    Chuang, Eleanore; Lim, Eunjung; Milne, Cris; Zhu, Xuemei; Agsalda, Melissa; Killeen, Jeffrey; Miller, F DeWolfe; Hernandez, Brenda Y.; Shiramizu, Bruce

    2016-01-01

    Objective HIV-Seropositive patients have higher risk of HPV infection even on anti-retroviral therapy. Infection with high-risk HPV genotypes can cause dysplasia leading to cancer. This study assessed HPV at different anatomical sites in HIV-seropositive individuals and factors associated with anal squamous intraepithelial lesions (ASIL). Methods Specimens were obtained from multiple anatomical sites for each participant in conjunction with routine screening for anal dysplasia. Female specimens included cervical and anal cytologies and oral wash. Male specimens included anal cytologies, oral wash, and exfoliated cells from penile head, penile shaft, scrotum, and from uncircumcised subjects, inner foreskin. Demographic and clinical characteristics were recorded. Following DNA extraction, HIV DNA copy was assessed by qPCR; HPV was genotyped. Statistical analyses included calculation of odds ratios (OR) and 95% confidence intervals (CI), t-tests or Mann-Whitney tests. Results Males were more likely to have ASIL: 29/50 (58%) compared to 1/11 females (9%) (OR=13.81, 95% CI: 1.64–116.32). HPV 6 or 11 in anal specimens was significantly associated with ASIL (OR= 6.29, 95% CI: 1.49–26.44). Number of HPV genotypes in anal specimens was also significant: ASIL+ (3.4 ± 3.1) versus ASIL− (1.6 ± 3.1) (p=0.009). Among 44 males, HPV was detected from at least one anatomical site for 33 participants (75%): 27 anus (61%), 19 oral wash (44%), 17 penile shaft (39%), 11 scrotum (26%), 10 penile head (23%), 0 foreskin. Detection of HPV in penile shaft specimens was significantly associated with ASIL (OR=6.79, 95% CI: 1.57–29.36) as was number of HPV genotypes in penile shaft specimens: ASIL+ (2.4 ± 4.0) versus ASIL− (0.6 ± 1.7) (p=0.025). Only 1/11 females had ASIL; only 1/11 females had cervical dysplasia: OR was not estimable due to small numbers. Conclusions Males were more prone to ASIL than females. HPV at anal as well as non-anal sites may be indicative of ASIL. PMID

  4. Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

    PubMed

    Kwon, Inchan; Choi, Eun Sil

    2016-01-01

    Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

  5. Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

    PubMed Central

    Kwon, Inchan; Choi, Eun Sil

    2016-01-01

    Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation. PMID:27028506

  6. The controlled relay of multiple protons required at the active site of nitrogenase.

    PubMed

    Dance, Ian

    2012-07-07

    The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

  7. Inclusion of multiple fragment types in the Site Identification by Ligand Competitive Saturation (SILCS) approach

    PubMed Central

    Raman, E. Prabhu; Yu, Wenbo; Lakkaraju, Sirish K.; MacKerell, Alexander D.

    2014-01-01

    The Site Identification by Ligand Competitive Saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing Molecular Dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors, and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple, diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced Ligand Grid Free Energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show some, but not all experimental trends are predicted, and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed. PMID:24245913

  8. Fatigue analysis of multiple site damage at a row of holes in a wide panel

    NASA Technical Reports Server (NTRS)

    Buhler, Kimberley; Grandt, Alten F., Jr.; Moukawsher, E. J.

    1994-01-01

    This paper is concerned with predicting the fatigue life of unstiffened panels which contain multiple site damage (MSD). The initial damage consists of through-the-thickness cracks emanating from a row of holes in the center of a finite width panel. A fracture mechanics analysis has been developed to predict the growth, interaction, and coalescence of the various cracks which propagate in the panel. A strain-life analysis incorporating Neuber's rule for notches, and Miner's rule for cumulative damage, is also employed to predict crack initiation for holes without initial cracking. This analysis is compared with the results of a series of fatigue tests on 2024-T3 aluminum panels, and is shown to do an excellent job of predicting the influence of MSD on the fatigue life of nine inch wide specimens. Having established confidence in the ability to analyze the influence of MSD on fatigue life, a parametric study is conducted to examine the influence of various MSD scenarios in an unstiffened panel. The numerical study considered 135 cases in all, with the parametric variables being the applied cyclic stress level, the lead crack geometry, and the number and location of MSD cracks. The numerical analysis provides details for the manner in which lead cracks and MSD cracks grow and coalesce leading to final failure. The results indicate that MSD located adjacent to lead cracks is the most damaging configuration, while for cases without lead cracks, MSD clusters which are not separated by uncracked holes are most damaging.

  9. Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein.

    PubMed

    Dee, Derek R; Gupta, Amar Nath; Anikovskiy, Max; Sosova, Iveta; Grandi, Elena; Rivera, Laura; Vincent, Abhilash; Brigley, Angela M; Petersen, Nils O; Woodside, Michael T

    2012-06-01

    The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90-232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al(3+)-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al(3+), Zn(2+), and free-base). Buffer ionic strength also affected the binding, with K(d) increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4-5 PcTS molecules per PrP molecule.

  10. Suppression of 5' splice-sites through multiple exonic motifs by hnRNP L.

    PubMed

    Loh, Tiing Jen; Choi, Namjeong; Moon, Heegyum; Jang, Ha Na; Liu, Yongchao; Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2017-03-01

    Selection of 5' splice-sites (5'SS) in alternative splicing plays an important role in gene regulation. Although regulatory mechanisms of heterogeneous nuclear ribonucleoprotein L (hnRNP L), a well-known splicing regulatory protein, have been studied in a substantial level, its role in 5'SS selection is not thoroughly defined. By using a KLF6 pre-mRNA alternative splicing model, we demonstrate in this report that hnRNP L inhibits proximal 5'SS but promotes two consecutive distal 5'SS splicing, antagonizing SRSF1 roles in KLF6 pre-mRNA splicing. In addition, three consecutive CA-rich sequences in a CA cassette immediately upstream of the proximal 5'SS are all required for hnRNP L functions. Importantly, the CA-cassette locations on the proximal exon do not affect hnRNP L roles. We further show that the proximal 5'SS but not the two distal 5'SSs are essential for hnRNP L activities. Notably, in a Bcl-x pre-mRNA model that contains two alternative 5'SS but includes CA-rich elements at distal exon, we demonstrate that hnRNP L also suppresses nearby 5'SS activation. Taken together, we conclude that hnRNP L suppresses 5'SS selection through multiple exonic motifs.

  11. Using level data to estimate inflow hydrographs in ungauged sites of multiple reach systems

    NASA Astrophysics Data System (ADS)

    D'Oria, M.; Mignosa, P.; Tanda, M.

    2013-12-01

    Considering a natural river system, usually only few sites are monitored and equipped to measure discharge over time. However, the knowledge of the discharge hydrograph in a specific station is important for many purposes: calibration of rainfall-runoff model, building of new structures, flood frequency analysis, water resource management, etc.. In addition, continuous direct measurement of discharges in open channels can be impossible and in any way difficult to assess. Therefore, most frequently, only level gauges are used for monitoring the system. In this work we propose a methodology to estimate a flow hydrograph in completely ungauged upstream sites of multiple reach systems by means of level data available at monitored stations downstream. In particular, a Bayesian approach is proposed to solve this kind of inverse problem. Prior information, in terms of geostatistical models and tools, is used to represent the structure of the unknowns and to regularize the solution. The methodology requires a forward hydraulic model of the considered river system able to reproduce the routing process and to account for all the reach characteristics: roughness, bed slope, cross sections, confluences, structures, etc.. In this work, the forward model is the widely known HEC-RAS river analysis system. The methodology has been tested through synthetic examples of river confluences, that differ in the available water level data, in the boundary conditions and in the number of the estimated inflow time series. Known inflow time-series were routed downstream by means of HEC-RAS to obtain the downstream stage hydrographs used as observations to test the reverse procedure. In almost all cases, the observed water levels were corrupted with random errors to highlight the reliability of the methodology in preventing instabilities and overfitting. The procedure has been also tested on a real case study of a river confluence located at the city of Parma (Italy) to assess the tributary

  12. Site and size of multiple sclerosis lesions predict enhanced or decreased female orgasmic function.

    PubMed

    Winder, Klemens; Seifert, Frank; Koehn, Julia; Deutsch, Martina; Engelhorn, Tobias; Dörfler, Arnd; Lee, De-Hyung; Linker, Ralf A; Hilz, Max J

    2015-12-01

    Neuroimaging identified brain areas involved in female orgasm. In women with multiple sclerosis (MS), associations between orgasmic function and the site and size of MS-related magnetic resonance imaging (MRI) changes are undetermined. This study intended to correlate MS-associated cerebral lesion load and location with clinical scores of female orgasmic function. In 50 women with MS (mean age 37.0 ± 9.9 years), we assessed Female Sexual Function Index (FSFI) scores for orgasmic frequency, difficulty and satisfaction. We determined disease duration, Expanded Disability Status Scale (EDSS) scores, and cerebral MS-lesion load and location using T2-weighed 1.5 T MRIs. We correlated FSFI scores for orgasm with patient age, disease duration, EDSS scores, and cerebral MS-lesion load (Spearman rank correlation; significance: p < 0.05). FSFI scores for orgasm correlated inversely with MS-lesion load in the left temporal periventricular white matter and right middle-inferior occipital area, but directly with MS-lesion load in the right frontal primary motor cortex, left prefrontal/inferior frontal cortex, right amygdala, left temporal middle-inferior and fusiform areas, and midbrain. FSFI scores for orgasm did not correlate with patient age, disease duration and EDSS scores (p > 0.05). In conclusion, our results indicate that MS-lesions in left temporal periventricular and right visual association areas deteriorate orgasmic function. In contrast, direct correlations between frontotemporal or midbrain lesions and higher FSFI scores, indicating enhanced or disinhibited orgasmic function, suggest that these brain regions normally buffer orgasmic responses. Moreover, our results indicate that orgasmic dysfunction in women with MS evolves independently of disease duration and physical disability.

  13. Analysis of persistence of human papillomavirus infection in men evaluated by sampling multiple genital sites.

    PubMed

    Capra, G; Nyitray, A G; Lu, B; Perino, A; Marci, R; Schillaci, R; Matranga, D; Firenze, A; Caleca, M; Bellavia, C; Guarneri, F; Giuliano, A; Giovannelli, L

    2015-11-01

    Although human papillomavirus (HPV) infection has been studied extensively in women, data on male infection are limited. The purpose of this study was to investigate persistence of HPV infection at multiple genital sites in men and to define potential associations with socio-behavioural characteristics. Penile, urethral and seminal specimens were tested by the INNO-LiPA HPV system (Innogenetics) and a PCR assay. Persistence was defined as the detection of same HPV type at ≥ 2 consecutive visits. The Kaplan-Meier method and the log-rank test were applied to estimate the likelihood of persistence. A total of 50 men (median age: 33 years) were followed for a median of 14.7 months. Altogether, 49%, 36%, 26% and 11% of baseline HPV-positive men had 6-, 12-, 18- and 24-month persistent infection with any HPV type, respectively. The 6-, 12- and 18- month persistence was more common for oncogenic HPV infections; 24-month persistence was similar. The median duration of persistence was 21.7 months for any HPV. The median duration of persistence for any HPV type was significantly longer in the penile sample (22.5 months, 95% CI: 18.3-26.7) than the semen sample (15.3 months, 95% CI: 14.5-16.1). Over a third of type-specific HPV infections in men remained persistent over a 24-month period. The median duration of HPV infection was longer in penile samples compared to seminal samples. As being increasing the attention of HPV vaccination as a potential preventive approach also for men, it is imperative to obtain additional insight on natural history of HPV infection in men, particularly as far as incidence and duration are concerned.

  14. 15 CFR 921.33 - Boundary changes, amendments to the management plan, and addition of multiple-site components.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... management plan, and addition of multiple-site components. (a) Changes in the boundary of a Reserve and major changes to the final management plan, including state laws or regulations promulgated specifically for the... management plan change. Changes in the boundary of a Reserve involving the acquisition of properties...

  15. 15 CFR 921.33 - Boundary changes, amendments to the management plan, and addition of multiple-site components.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... management plan, and addition of multiple-site components. (a) Changes in the boundary of a Reserve and major changes to the final management plan, including state laws or regulations promulgated specifically for the... management plan change. Changes in the boundary of a Reserve involving the acquisition of properties...

  16. 15 CFR 921.33 - Boundary changes, amendments to the management plan, and addition of multiple-site components.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... management plan, and addition of multiple-site components. (a) Changes in the boundary of a Reserve and major changes to the final management plan, including state laws or regulations promulgated specifically for the... management plan change. Changes in the boundary of a Reserve involving the acquisition of properties not...

  17. 15 CFR 921.33 - Boundary changes, amendments to the management plan, and addition of multiple-site components.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... management plan, and addition of multiple-site components. (a) Changes in the boundary of a Reserve and major changes to the final management plan, including state laws or regulations promulgated specifically for the... management plan change. Changes in the boundary of a Reserve involving the acquisition of properties not...

  18. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    SciTech Connect

    James, I.F.; Goldstein, A.

    1984-05-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, (/sup 3/H) dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for (/sup 3/H) (D-Ala2, D-Leu5)enkephalin and (3H)ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites.

  19. Prevalence of Osteoporosis and Low Bone Mass in Older Chinese Population Based on Bone Mineral Density at Multiple Skeletal Sites

    PubMed Central

    Lu, Yi-Chien; Lin, Ying Chin; Lin, Yen-Kuang; Liu, Yi-Jui; Chang, Kwang-Hwa; Chieng, Poon-Ung; Chan, Wing P.

    2016-01-01

    Diagnosis of osteoporosis is based on bone mineral density (BMD) measurement, which is site dependent and commonly discordant between measurement sites. We aimed to determine the prevalence of osteoporosis diagnosed based on BMD T-scores measured by dual-energy x-ray absorptiometry (DXA) at different sites: the lumbar spine (LS) alone, femoral neck (FN) alone, or both. A total of 1712 women and 2028 men with LS and FN BMD measurements were enrolled. Over 50% discordance was found between osteoporosis classifications based on T-scores measured at the LS and FN. Use of the lowest T-scores measured at both the LS and right and left FN (rather than one site) significantly increased the prevalence of osteoporosis from 4.03 to 10.75% in postmenopausal women and 1.82 to 4.29% in men aged ≧50 years (p < 0.001). The trends of overall and age-adjusted prevalence of osteoporosis were similar in women and men. Osteoporosis was diagnosed at a higher rate if the USA reference rather than the Asia reference was used to calculate the T-score (26.64% vs. 10.75%). In conclusion, diagnosis based on the lowest T-score from multiple site BMD measurement can increase the prevalence of osteoporosis, demonstrating the higher sensitivity of the multiple site measurement strategy. PMID:27143609

  20. Prevalence of Osteoporosis and Low Bone Mass in Older Chinese Population Based on Bone Mineral Density at Multiple Skeletal Sites.

    PubMed

    Lu, Yi-Chien; Lin, Ying Chin; Lin, Yen-Kuang; Liu, Yi-Jui; Chang, Kwang-Hwa; Chieng, Poon-Ung; Chan, Wing P

    2016-05-04

    Diagnosis of osteoporosis is based on bone mineral density (BMD) measurement, which is site dependent and commonly discordant between measurement sites. We aimed to determine the prevalence of osteoporosis diagnosed based on BMD T-scores measured by dual-energy x-ray absorptiometry (DXA) at different sites: the lumbar spine (LS) alone, femoral neck (FN) alone, or both. A total of 1712 women and 2028 men with LS and FN BMD measurements were enrolled. Over 50% discordance was found between osteoporosis classifications based on T-scores measured at the LS and FN. Use of the lowest T-scores measured at both the LS and right and left FN (rather than one site) significantly increased the prevalence of osteoporosis from 4.03 to 10.75% in postmenopausal women and 1.82 to 4.29% in men aged ≧50 years (p < 0.001). The trends of overall and age-adjusted prevalence of osteoporosis were similar in women and men. Osteoporosis was diagnosed at a higher rate if the USA reference rather than the Asia reference was used to calculate the T-score (26.64% vs. 10.75%). In conclusion, diagnosis based on the lowest T-score from multiple site BMD measurement can increase the prevalence of osteoporosis, demonstrating the higher sensitivity of the multiple site measurement strategy.

  1. Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

    PubMed Central

    Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

    1994-01-01

    Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

  2. Studies of polar mesosphere summer echoes at multiple sites using calibrated radars

    NASA Astrophysics Data System (ADS)

    Swarnalingam, Nimalan

    A 51.5 MHz VHF radar system, located at Resolute Bay in northern Canada has been monitoring Polar Mesosphere Summer Echoes (PMSE) since 1997. This is currently the northernmost radar used to monitor PMSE in the North American sector. It has been recording a lower level of PMSE activity in this region, compared with the PMSE activity recorded by other high latitudes radars operating at similar frequencies. To investigate the reason for this, the radar system has been absolutely calibrated by means of cosmic sky noise variations along with the help of a calibrated noise source. A general theory is developed in such a way that the calibration procedure can be directly applied to any MST radar system anywhere in the world, provided the sky noise variations are known. PMSE backscatter cross-sections for the Resolute Bay radar are estimated, converted into an equivalent electron density structure function constant, C2N , and compared with three other radars located at Yellowknife and Andenes during simultaneous measurements. It was found that the estimated C2N for Resolute Bay is at least an order of magnitude lower than the estimated values at the other two locations. This leads us to conclude that the reason why PMSE strengths at Resolute Bay are weak compared with other sites is geophysical in origin, and not due to instrumental effects. The seasonal and diurnal variations of PMSE at Resolute Bay, and their connection to cold mesopause temperatures, are investigated on a long term basis, and compared with the observations at Andenes on a short term basis. Although the PMSE season is relatively short at Resolute Bay, the PMSE temporal variation pattern agrees with Andenes for at least one season. On the other hand, the two sites show a completely different picture for diurnal variations. While PMSE at Andenes show a clear diurnal variation, PMSE at Resolute Bay are modulated by high frequency oscillations. For the first time, a group of near identical SKiYMET meteor

  3. Correlation between reticle- and wafer-CD difference of multiple 28nm reticle-sites

    NASA Astrophysics Data System (ADS)

    Ning, Guoxiang; Richter, Frank; Thamm, Thomas; Ackmann, Paul; Staples, Marc; Weisbuch, Francois; Kurth, Karin; Schenker, Joerg; Leschok, Andre; GN, Fang Hong

    2012-11-01

    Reticle critical dimension uniformity (CDU) is an important criterion for the qualification of mask layer processes. Normally, the smaller the three sigma value of reticle CDU is, the better is the reticle CDU performance. For qualification of mask processes, the mask layers to be qualified should have a comparable reticle CDU compared to the process of record (POR) mask layers. Because the reticle critical dimension (CD) measurement is based on algorithms like "middle side lobe measurement", evaluation of the reticle CD-values can not reflect aspects like the sidewall angle of the reticle and variation in corner rounding which may be critical for 45nm technology nodes (and below). All involved tools and processes contribute to the wafer intra-field CDU (scanner, track, reticle, metrology). Normally, the reticle contribution to the wafer CDU should be as small as possible. In order to reduce the process contributions to the wafer intra-field CDU during the mask qualification process, the same toolset (exposure tool, metrology tool) should be applied as for the POR. Out of the results of these investigations the correlation between wafer measurement to target (MTT) and reticle MTT can be obtained in order to accurately qualify the CDU performance of the mask processes. We will demonstrate the correlation between reticle MTT and wafer MTT by use of multiple mask processes and alternative mask blank materials. We will investigate the results of four process-layers looking at advanced binary maskblank material from two different suppliers (moreover the results of a 2X-via layer as an example for a phase-shift maskblank is discussed). Objective of this article is to demonstrate the distribution between reticle MTT and wafer MTT as a qualification criterion for mask processes. The correlation between wafer CD-difference and reticle CD-difference of these mask processes are demonstrated by having performed investigations of dense features of different 28nmtechnology

  4. Clinical implications of single- versus multiple-site keloid disorder: a retrospective study in an Asian population.

    PubMed

    Park, Tae Hwan; Park, Ji Hae; Tirgan, Michael H; Halim, Ahmad Sukari; Chang, Choong Hyun

    2015-02-01

    There is strong evidence of genetic susceptibility in individuals with keloid disorder. The purpose of this cross-sectional study was to determine the clinical relevance of our proposed variables on the multiplicity of keloids by further investigating the presence of other keloids and a family history. This was a retrospective review, using institutional review board-approved questionnaires, of patients with keloids who were seen at Kangbuk Samsung Hospital between December 2002 and February 2010. Eight hundred sixty-eight patients were included in our study. Comparisons between the 2 groups were made using Mann-Whitney tests for continuous variables and χ2 tests for categorical variables. In our patient group, younger age of onset and the presence of family history were significantly associated with the occurrence of keloids at multiple sites. The locations of extra-auricular keloids, in order of frequency, included the shoulder; anterior chest, including the breasts; deltoid; trunk and pubic area; upper extremities; lower extremities; and other sites. As compared to secondary keloids, primary keloids were significantly associated with both a lower degree of recurrence and the presence of other keloids. The presence or absence of family history was significantly associated with the presence or absence of other keloids and primary or secondary keloids. Keloid disorder is one of the most frustrating problems in wound healing and advances in our understanding of the differences of occurrence at a single site versus multiple sites might help in understanding pathogenesis and improving treatment.

  5. Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

    PubMed Central

    Peng, Yu-Cai; Acheson, Nicholas H.

    1998-01-01

    Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts. PMID:9696829

  6. Multiple Ca2+ Binding Sites in the Extracellular Domain of Ca2+-Sensing Receptor Corresponding to Cooperative Ca2+ Response†

    PubMed Central

    Huang, Yun; Zhou, Yubin; Castiblanco, Adriana; Yang, Wei; Brown, Edward M.; Yang, Jenny J.

    2009-01-01

    A small change in the extracellular Ca2+ concentration ([Ca2+]o) integrates cell signaling responses in multiple cellular and tissue networks and functions via activation of Ca2+-sensing receptors (CaSR). Mainly through binding of Ca2+ to the large extracellular domain (ECD) of the dimeric CaSR, intracellular Ca2+ responses are highly cooperative with an apparent Hill coefficient ranging from 2 to 4. We have previously reported the identification of two continuous putative Ca2+-binding sites by grafting CaSR-derived, Ca2+-binding peptides to a scaffold protein, CD2, that does not bind Ca2+. In this paper, we predict more potential non-continuous Ca2+-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains 2 to 3 predicted Ca2+-binding sites. This approach enables us to further understand the mechanisms underlying the binding of multiple metal ions to extended polypeptides derived from within the ECD of the CaSR, which would be anticipated to more closely mimic the structure of the native CaSR ECD. Tb3+-luminescence energy transfer, ANS fluorescence, and NMR studies show biphasic metal-binding components and Ca2+-dependent conformational changes in these subdomains. Removing the predicted Ca2+-binding ligands in site 1 and site 3 abolishes the first binding step and second binding step, respectively. Studies on these subdomains suggest the existence of multiple metal-binding sites and metal-induced conformational changes that might be responsible for switching on/off the CaSR by transition between its open inactive form and closed active form. PMID:19102677

  7. Transient Protein-Protein Interaction of the SH3-Peptide Complex via Closely Located Multiple Binding Sites

    PubMed Central

    Hahn, Seungsoo; Kim, Dongsup

    2012-01-01

    Protein-protein interactions play an essential role in cellular processes. Certain proteins form stable complexes with their partner proteins, whereas others function by forming transient complexes. The conventional protein-protein interaction model describes an interaction between two proteins under the assumption that a protein binds to its partner protein through a single binding site. In this study, we improved the conventional interaction model by developing a Multiple-Site (MS) model in which a protein binds to its partner protein through closely located multiple binding sites on a surface of the partner protein by transiently docking at each binding site with individual binding free energies. To test this model, we used the protein-protein interaction mediated by Src homology 3 (SH3) domains. SH3 domains recognize their partners via a weak, transient interaction and are therefore promiscuous in nature. Because the MS model requires large amounts of data compared with the conventional interaction model, we used experimental data from the positionally addressable syntheses of peptides on cellulose membranes (SPOT-synthesis) technique. From the analysis of the experimental data, individual binding free energies for each binding site of peptides were extracted. A comparison of the individual binding free energies from the analysis with those from atomistic force fields gave a correlation coefficient of 0.66. Furthermore, application of the MS model to 10 SH3 domains lowers the prediction error by up to 9% compared with the conventional interaction model. This improvement in prediction originates from a more realistic description of complex formation than the conventional interaction model. The results suggested that, in many cases, SH3 domains increased the protein complex population through multiple binding sites of their partner proteins. Our study indicates that the consideration of general complex formation is important for the accurate description of

  8. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

  9. NATURAL ATTENUATION OF CHLORINATED SOLVENTS AT MULTIPLE AIR FORCE BASE DEMONSTRATION SITES

    EPA Science Inventory

    Natural attenuation treatability studies(TSs) were conducted at 14 US Air Force bases. Only sites where biodegradation of CAHs was suspected were selected for the study. The major initiative was to evaluate the effectiveness of monitored natural attenuation(MNA) at sites contam...

  10. NATURAL ATTENUATION OF CHLORINATED SOLVENTS AT MULTIPLE AIR FORCE BASE DEMONSTRATION SITES

    EPA Science Inventory

    Natural attenuation treatability studies(TSs) were conducted at 14 US Air Force bases. Only sites where biodegradation of CAHs was suspected were selected for the study. The major initiative was to evaluate the effectiveness of monitored natural attenuation(MNA) at sites contam...

  11. Prediction of P53 Mutants (Multiple Sites) Transcriptional Activity Based on Structural (2D&3D) Properties

    PubMed Central

    Geetha Ramani, R.; Jacob, Shomona Gracia

    2013-01-01

    Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site) p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis. PMID:23468845

  12. National Register of Historic Places multiple property documentation form -- Historic, archaeological, and traditional cultural properties of the Hanford Site, Washington

    SciTech Connect

    Nickens, P.R.

    1997-08-01

    The US Department of Energy`s Hanford Site encompasses an area of 560 square miles on the Columbia River in southeastern Washington. Since 1943, the Hanford Site has existed as a protected area for activities primarily related to the production of radioactive materials for national defense uses. For cultural resources on the Hanford Site, establishment of the nuclear reservation as a high security area, with public access restricted, has resulted in a well-protected status, although no deliberate resource protection measures were in effect to mitigate effects of facilities construction and associated activities. Thus, the Hanford Site contains an extensive record of aboriginal archaeological sites and Native American cultural properties, along with pre-Hanford Euro-American sites (primarily archaeological in nature with the removal of most pre-1943 structures), and a considerable number of Manhattan Project/Cold War era buildings and structures. The recent mission change from production to clean up and disposal of DOE lands created a critical need for development and implementation of new and different cultural resource management strategies. DOE-RL has undertaken a preservation planning effort for the Hanford Site. The intent of this Plan is to enable DOE-RL to organize data and develop goals, objectives, and priorities for the identification, evaluation, registration, protection, preservation, and enhancement of the Site`s historical and cultural properties. Decisions made about the identification, evaluation, registration and treatment of historic properties are most aptly made when relationships between individual properties and other similar properties are considered. The historic context and the multiple property documentation (NTD) process provides DOE-RL the organizational framework for these decisions. Once significant patterns are identified, contexts developed, and expected properties are defined, the NTD process provides the foundation for future

  13. Analytical reduction of combinatorial complexity arising from multiple protein modification sites

    PubMed Central

    Birtwistle, Marc R.

    2015-01-01

    Combinatorial complexity is a major obstacle to ordinary differential equation (ODE) modelling of biochemical networks. For example, a protein with 10 sites that can each be unphosphorylated, phosphorylated or bound to adaptor protein requires 310 ODEs. This problem is often dealt with by making ad hoc assumptions which have unclear validity and disallow modelling of site-specific dynamics. Such site-specific dynamics, however, are important in many biological systems. We show here that for a common biological situation where adaptors bind modified sites, binding is slow relative to modification/demodification, and binding to one modified site hinders binding to other sites, for a protein with n modification sites and m adaptor proteins the number of ODEs needed to simulate the site-specific dynamics of biologically relevant, lumped bound adaptor states is independent of the number of modification sites and equal to m + 1, giving a significant reduction in system size. These considerations can be relaxed considerably while retaining reasonably accurate descriptions of the true system dynamics. We apply the theory to model, using only 11 ODEs, the dynamics of ligand-induced phosphorylation of nine tyrosines on epidermal growth factor receptor (EGFR) and primary recruitment of six signalling proteins (Grb2, PI3K, PLCγ1, SHP2, RasA1 and Shc1). The model quantitatively accounts for experimentally determined site-specific phosphorylation and dephosphorylation rates, differential affinities of binding proteins for the phosphorylated sites and binding protein expression levels. Analysis suggests that local concentration of site-specific phosphatases such as SHP2 in membrane subdomains by a factor of approximately 107 is critical for effective site-specific regulation. We further show how our framework can be extended with minimal effort to consider binding cooperativity between Grb2 and c-Cbl, which is important for receptor trafficking. Our theory has potentially broad

  14. Metabolic profile biomarkers of metal contamination in a sentinel terrestrial species are applicable across multiple sites.

    PubMed

    Bundy, Jacob G; Keun, Hector C; Sidhu, Jasmin K; Spurgeon, David J; Svendsen, Claus; Kille, Peter; Morgan, A John

    2007-06-15

    In this study, we addressed the question of whether an omic approach could genuinely be useful for biomarker profile analysis across different field sites with different physicochemical characteristics. We collected earthworms (Lumbricus rubellus) from seven sites with very different levels of metal contamination and prevailing soil type and analyzed tissue extracts by 1H nuclear magnetic resonance spectroscopy. Pattern recognition analysis of the data showed that both site- and contaminant-specific effects on the metabolic profiles could be discerned. Zinc was identified as the probable major contaminant causing a metabolic change in the earthworms. Individual sites could be resolved on the basis of NMR spectral profiles by principal component analysis; these site differences may also have been caused by additional abiotic factors such as soil pH. Despite an inevitable degree of confounding between site and contaminant concentrations, it was possible to identify metabolites which were correlated with zinc across all different sites. This study therefore acts as a proof of principle for the use of NMR-based metabolic profiling as a diagnostic tool for ecotoxicological research in polluted field soils.

  15. Fast optical measurement of membrane potential changes at multiple sites on an individual nerve cell.

    PubMed

    Zecević, D; Antić, S

    1998-03-01

    individual neuron: (1) the direction and the velocity of action potential propagation in different neuronal processes in the neuropile was determined; and (2) the interaction of two independent action potentials (spike collision) was monitored directly in a neurite in the neuropile; (3) it was demonstrated that several action potentials are initiated in the same neuron at different sites (multiple spike trigger zones) by a single stimulus; (4) the exact location and the size of one of the remote spike trigger zones was determined; (5) the spread of passive subthreshold signals was followed in the neurites in the neuropile. This kind of information was not previously available. Preliminary experiments on vertebrate neurons indicate partial success in the effort to use intracellularly applied voltage-sensitive dyes to record from neurons in a mammalian brain slice preparation. The results suggest that, with further improvements, it may be possible to follow optically synaptic integration and spike conduction in the dendrites of vertebrate nerve cells. The main impact of these results is a demonstration of a new way of analysing how individual neurons are functionally organized. Limitations and prospects for the further refinement of the technique are discussed mostly in terms of the signal-to-noise ratio; both improvements in the apparatus and design of more sensitive dyes are addressed.

  16. The genealogy of sequences containing multiple sites subject to strong selection in a subdivided population.

    PubMed

    Nordborg, Magnus; Innan, Hideki

    2003-03-01

    A stochastic model for the genealogy of a sample of recombining sequences containing one or more sites subject to selection in a subdivided population is described. Selection is incorporated by dividing the population into allelic classes and then conditioning on the past sizes of these classes. The past allele frequencies at the selected sites are thus treated as parameters rather than as random variables. The purpose of the model is not to investigate the dynamics of selection, but to investigate effects of linkage to the selected sites on the genealogy of the surrounding chromosomal region. This approach is useful for modeling strong selection, when it is natural to parameterize the past allele frequencies at the selected sites. Several models of strong balancing selection are used as examples, and the effects on the pattern of neutral polymorphism in the chromosomal region are discussed. We focus in particular on the statistical power to detect balancing selection when it is present.

  17. Source apportionment of particulate organic matter using infrared spectra at multiple IMPROVE sites

    NASA Astrophysics Data System (ADS)

    Kuzmiakova, A.; Dillner, A. M.; Takahama, S.

    2016-12-01

    As organic aerosol is a dominant contributor to air pollution and radiative forcing in many regions in the United States, characterizing its composition and apportioning the organic mass to its major sources provides insight into atmospheric processes and guidance for decreasing its abundance. National networks, such as Interagency Monitoring of Protected Visual Environment (IMPROVE), provide multi-site and multi-year particulate matter samples useful for evaluating sources over all four seasons. To this end, our study focuses on apportioning the particulate organic matter (OM) to specific anthropogenic and biological processes from year-long infrared aerosol measurements collected at six IMPROVE sites (five national park sites and one urban site) during 2011. Pooling these organic aerosol samples into one dataset, we apply factor and cluster analyses to extract four chemical factors (two dominated by processed emissions, one dominated by hydroxyl groups, and one by hydrocarbons) and ascribe each factor to a specific source depending on the site and season. We also present a method to characterize measurement uncertainty in infrared instrumental analysis and investigate sensitivity analysis in generated factors. In Phoenix (the urban site) we find the majority (80-95%) of the OM consisted of anthropogenic activities, such as traffic emissions, fossil fuel combustion (both all year long), and residential wood burning (fall to winter). Mineral dust emissions accounted for the rest of OM (5-20%). At the National Park sites the OM concentration was lower on average and consisted of marine and dust aerosols, summertime biomass burning and biogenic aerosols, processed fossil fuel combustion, and emissions from ships and oil refineries. Our study highlights the potential for further site-specific or multi-year aerosol characterization in the context of a long-term atmospheric sampling program to quantify sources of organic particles impacting air quality, aid in policy

  18. 43 CFR 2806.38 - Can I combine multiple grants or leases for facilities located on one site into a single grant or...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 2 2011-10-01 2011-10-01 false Can I combine multiple grants or leases for facilities located on one site into a single grant or lease? 2806.38 Section 2806.38 Public Lands... Communication Site Rights-Of-Way § 2806.38 Can I combine multiple grants or leases for facilities located on...

  19. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase

    PubMed Central

    Jacobs, Dave; Glossip, Danielle; Xing, Heming; Muslin, Anthony J.; Kornfeld, Kerry

    1999-01-01

    MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo. PMID:9925641

  20. Theory and simulation of chain molecules with multiple bonding sites in an associating solvent

    NASA Astrophysics Data System (ADS)

    García-Cuéllar, Alejandro J.; Chapman, Walter G.

    2011-08-01

    Although polyethylene oxide (PEO) is soluble in water, polymethylene oxide (PMO) is not, even though PMO has more association sites. Some suggest this is due to orientation effects in the water hydrogen-bond network. A simulation and theory study of the effect of bonding site density on thermodynamic properties and extent of bonding of a linear flexible chain in a hydrogen-bonding solvent is performed. Predictions from Wertheim's theory are compared against simulation results. Thermodynamic properties and extent of bonding were obtained. The solvent molecules are modeled as hard spheres with four association sites in a tetrahedral arrangement. The chains are flexible and consist of six tangent segments of hard spheres with bonding sites that interact with the solvent molecules. A solvent molecule can also form a bond with a second solvent molecule. The association interaction is modeled with an orientation-dependent square-well. The total number of bonding sites on each chain is varied and the effects studied. This is another test of the theory for the case of mixtures of associating molecules of different sizes. The Metropolis Monte Carlo technique was chosen to perform simulations in the canonical and isothermal-isobaric ensembles. Good agreement was found between theory and simulation.

  1. Neural networks for determining protein specificity and multiple alignment of binding sites

    SciTech Connect

    Heumann, J.M.; Lapedes, A.S.; Stormo, G.D.

    1994-12-31

    We use a quantitative definition of specificity to develop a neural network for the identification of common protein binding sites in a collection of unaligned DNA fragments. We demonstrate the equivalence of the method to maximizing Information Content of the aligned sites when simple models of the binding energy and the genome are employed. The network method subsumes those simple models and is capable of working with more complicated ones. This is demonstrated using a Markov model of the E. coli genome and a sampling method to approximate the partition function. A variation of Gibbs sampling aids in avoiding local minima.

  2. Initial atmospheric-dispersion modeling in support of the multiple-site incineration study

    SciTech Connect

    Holton, G. A.; Little, C. A.; O'Donnell, F. R.; Etnier, E. L.; Travis, C. C.

    1982-04-01

    Several modeling series which estimate population exposure to stack emissions from incineration of hazardous organic materials at 22 commercial incinerator sites are presented. These modeling series can be divided into three groups. One group estimates long- and short-term atmospheric concentrations and population exposures for each of 22 sites. These modeling predictions can be used to assess chronic and acute exposure. The second group consists of sensitivity analyses which show the effect of changes in stack parameters on the number of people exposed to specified concentration levels. The third group compares concentration estimates of two atmospheric dispersion computer codes. Results of each modeling series are contained in the appendices of this report.

  3. Multiple transcription start sites for FOXP2 with varying cellular specificities.

    PubMed

    Schroeder, Diane I; Myers, Richard M

    2008-04-30

    FOXP2 is a forkhead transcription factor implicated in developmental verbal dyspraxia, a human speech and language disorder. FOXP2 is expressed in complex patterns during brain, lung, heart, and gut development and on into adulthood. Both the protein sequence and brain expression patterns are highly conserved through much of vertebrate evolution. FOXP2 also contains six ultraconserved regions in its introns, consistent with the idea that FOXP2 is tightly regulated. Previous evidence suggested the presence of three transcription start sites for FOXP2, and we sought to characterize them and determine their cell and tissue specificity. We used 5' RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) and RT-PCR to identify four transcription start sites for human FOXP2, the fourth being in a novel exon. Two of the transcription start sites, including the one in the novel exon, appear to be more cell line specific and lie in an area of remarkably high conservation. We propose that these two transcription start sites may be of great interest in future studies of FOXP2 regulation.

  4. NATURAL ATTENUATION OF FUEL HYDROCARBONS AT MULTIPLE AIR FORCE BASE DEMONSTRATION SITES

    EPA Science Inventory

    A major initiative to evaluate monitored natural attenuation(MNA) of ground water contaminated with fuel hydrocarbons began in June 1993 and continued through October 2000. During this time site characterization studies, both initial and follow-up, were conducted at 28 Air Forc...

  5. Heisenberg antiferromagnetic chain with multiple spin 1/2 particles of different flavors per site

    NASA Astrophysics Data System (ADS)

    Duki, Solomon F.; Yu, Yi-Kuo

    Motivated by the discoveries of quasi-1D magnetic systems, we studied a quantum mechanical spin lattice system consisting of a one-dimensional antiferromagnetic Heisenberg chain. In this system we considered M spin 1/2 particles of different flavors per site, and the low-lying states, ground state included, of the Hamiltonian was solved numerically using the exact diagonalization method for finite cluster sizes. We have also obtained the corresponding solutions for systems of the same chain length but with one spin M/2 particle per site. The low energy spectra of both systems are then compared. For M = 2 and M =3, our result shows that the two spin chain systems (one spin M/2 per site vs. M spin 1/2 of different flavors per site) have the same excitation spectra at low energy and the number of overlapped states increases as the size of the cluster increases. The observed overlap also indicates that low energy excitations of the M flavored spin 1/2 chain system selects the high spin states, effectively satisfying the Hund's Rule even though the system does not possess the orbital angular momentum. This work was supported by the Intramural Research Program of the National Library of Medicine at the National Institutes of Health.

  6. Biochemical study of multiple drug recognition sites on central benzodiazepine receptors

    SciTech Connect

    Trifiletti, R.R.

    1986-01-01

    The benzodiazepine receptor complex of mammalian brain possesses recognition sites which mediate (at least in part) the pharmacologic actions of the 1,4-benzodiazepines and barbiturates. Evidence is provided suggesting the existence of least seven distinct drug recognition sites on this complex. Interactions between the various recognition sites have been explored using radioligand binding techniques. This information is utilized to provide a comprehensive scheme for characterizing receptor-active drugs on an anxiolytic-anticonvulsant/proconvulsant continuum using radioligand binding techniques, as well as a comprehensive program for identifying potential endogenous receptor-active substances. Further evidence is provided here supporting the notion of benzodiazepine recognition site heterogeneity. Classical 1,4-benzodiazepines do not appear to differentiate two populations of benzodiazepine receptors in an equilibrium sense, but appear to do so in a kinetic sense. An apparent physical separation of the two receptor subtypes can be achieved by differential solubilization. The benzodiazepine binding subunit can be identified by photoaffinity labeling with the benzodiazepine agonist (/sup 3/H)flunitrazepan. Conditions for reproducible partial proteolytic mapping of (/sup 3/H)flunitrazepam photoaffinity labeled receptors are established. From these maps, it is concluded that there are probably no major differences in the primary sequence of the benzodiazepine binding subunit in various regions of the rat central nervous system.

  7. Assessment of multiple geophysical techniques for the characterization of municipal waste deposit sites

    NASA Astrophysics Data System (ADS)

    Gaël, Dumont; Tanguy, Robert; Nicolas, Marck; Frédéric, Nguyen

    2017-10-01

    In this study, we tested the ability of geophysical methods to characterize a large technical landfill installed in a former sand quarry. The geophysical surveys specifically aimed at delimitating the deposit site horizontal extension, at estimating its thickness and at characterizing the waste material composition (the moisture content in the present case). The site delimitation was conducted with electromagnetic (in-phase and out-of-phase) and magnetic (vertical gradient and total field) methods that clearly showed the transition between the waste deposit and the host formation. Regarding waste deposit thickness evaluation, electrical resistivity tomography appeared inefficient on this particularly thick deposit site. Thus, we propose a combination of horizontal to vertical noise spectral ratio (HVNSR) and multichannel analysis of the surface waves (MASW), which successfully determined the approximate waste deposit thickness in our test landfill. However, ERT appeared to be an appropriate tool to characterize the moisture content of the waste, which is of prior information for the organic waste biodegradation process. The global multi-scale and multi-method geophysical survey offers precious information for site rehabilitation studies, water content mitigation processes for enhanced biodegradation or landfill mining operation planning.

  8. Crosstalk between signaling pathways provided by single and multiple protein phosphorylation sites

    PubMed Central

    Nishi, Hafumi; Demir, Emek; Panchenko, Anna R.

    2014-01-01

    Cellular fate depends on the spatio-temporal separation and integration of signaling processes which can be provided by phosphorylation events. In this study we identify the crucial points in signaling crosstalk which can be triggered by discrete phosphorylation events on a single target protein. We integrated the data on individual human phosphosites with the evidence on their corresponding kinases, the functional consequences on phosphorylation on activity of the target protein and corresponding pathways. Our results show that there is a substantial fraction of phosphosites that can play critical roles in crosstalk between alternative or redundant pathways and regulatory outcome of phosphorylation can be linked to a type of phosphorylated residue. These regulatory phosphosites can serve as hubs in the signal flow and their functional roles are directly connected to their specific properties. Namely, phosphosites with similar regulatory functions are phosphorylated by the same kinases and participate in regulation of similar biochemical pathways. Such sites are more likely to cluster in sequence and space unlike sites with antagonistic outcomes of their phosphorylation on a target protein. In addition we found that in silico phosphorylation of sites with similar functional consequences have comparable outcomes on a target protein stability. An important role of phosphorylation sites in biological crosstalk is evident from the analysis of their evolutionary conservation. PMID:25451034

  9. NATURAL ATTENUATION OF FUEL HYDROCARBONS AT MULTIPLE AIR FORCE BASE DEMONSTRATION SITES

    EPA Science Inventory

    A major initiative to evaluate monitored natural attenuation(MNA) of ground water contaminated with fuel hydrocarbons began in June 1993 and continued through October 2000. During this time site characterization studies, both initial and follow-up, were conducted at 28 Air Forc...

  10. 12 CFR 1010.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...-determination required. 1010.15 Section 1010.15 Banks and Banking BUREAU OF CONSUMER FINANCIAL PROTECTION LAND..., where the developer either owns contiguous land or holds an option or other evidence of intent to acquire contiguous land which, when taken cumulatively, would or could result in one site of 100 or...

  11. 12 CFR 1010.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...-determination required. 1010.15 Section 1010.15 Banks and Banking BUREAU OF CONSUMER FINANCIAL PROTECTION LAND..., where the developer either owns contiguous land or holds an option or other evidence of intent to acquire contiguous land which, when taken cumulatively, would or could result in one site of 100 or...

  12. 12 CFR 1010.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...-determination required. 1010.15 Section 1010.15 Banks and Banking BUREAU OF CONSUMER FINANCIAL PROTECTION LAND..., where the developer either owns contiguous land or holds an option or other evidence of intent to acquire contiguous land which, when taken cumulatively, would or could result in one site of 100 or...

  13. Siting multiple conservation practices in a tile-drained watershed using LiDAR topographic data

    USDA-ARS?s Scientific Manuscript database

    Improving the effectiveness of conservation practices for achieving water quality goals will require a conservation planning with a watershed context. Conservation planning has traditionally been field or farm specific, and is usually aided by site specific information that includes topographic data...

  14. Multiple clusters of release sites formed by individual thalamic afferents onto cortical interneurons ensure reliable transmission.

    PubMed

    Bagnall, Martha W; Hull, Court; Bushong, Eric A; Ellisman, Mark H; Scanziani, Massimo

    2011-07-14

    Thalamic afferents supply the cortex with sensory information by contacting both excitatory neurons and inhibitory interneurons. Interestingly, thalamic contacts with interneurons constitute such a powerful synapse that even one afferent can fire interneurons, thereby driving feedforward inhibition. However, the spatial representation of this potent synapse on interneuron dendrites is poorly understood. Using Ca imaging and electron microscopy we show that an individual thalamic afferent forms multiple contacts with the interneuronal proximal dendritic arbor, preferentially near branch points. More contacts are correlated with larger amplitude synaptic responses. Each contact, consisting of a single bouton, can release up to seven vesicles simultaneously, resulting in graded and reliable Ca transients. Computational modeling indicates that the release of multiple vesicles at each contact minimally reduces the efficiency of the thalamic afferent in exciting the interneuron. This strategy preserves the spatial representation of thalamocortical inputs across the dendritic arbor over a wide range of release conditions.

  15. Multiple saxitoxin-binding sites in bullfrog muscle: tetrodotoxin-sensitive sodium channels and tetrodotoxin-insensitive sites of unknown function

    SciTech Connect

    Moczydlowski, E.; Mahar, J.; Ravindran, A.

    1988-02-01

    The possible presence of multiple sodium channel subtypes in bullfrog skeletal muscle was investigated in binding experiments with (/sup 3/H)saxitoxin and in single-channel studies using planar lipid bilayers. Two classes of (/sup 3/H)saxitoxin-binding sites were identified in membrane preparations. One class displayed a toxin specificity characteristic of voltage-dependent sodium channels: high affinity for saxitoxin (KD approximately equal to 0.5 nM), neosaxitoxin (KD approximately equal to 0.1 nM), and tetrodotoxin (KD approximately equal to 1.3 nM). A second class of membrane-associated binding sites exhibited high affinity for saxitoxin (KD approximately equal to 0.1 nM), lower affinity for neosaxitoxin (KD approximately equal to 25 nM), and complete insensitivity to tetrodotoxin at concentrations up to 32 microM. The first class corresponded to functional tetrodotoxin-sensitive sodium channels that could be incorporated and observed in planar bilayers in the presence of batrachotoxin. The unusual, tetrodotoxin-insensitive binding activity for (/sup 3/H)saxitoxin was also found at nM levels in the high speed supernatant of homogenized skeletal muscle without the addition of detergents. This soluble class of sites exhibited low affinity for neosaxitoxin (KD approximately equal to 60 nM) and a very slow dissociation rate of (/sup 3/H)saxitoxin (t0.5 approximately equal to 90 min), properties nearly identical to those of the tetrodotoxin-insensitive sites in membranes. The soluble saxitoxin-binding activity is also characterized by a more basic pH dependence and a complete lack of binding competition between saxitoxin and alkali cations. Bullfrog muscle appears to be a good tissue source for the purification of this soluble saxitoxin-binding protein.

  16. Maturation of the Infant Microbiome Community Structure and Function Across Multiple Body Sites and in Relation to Mode of Delivery

    PubMed Central

    Chu, Derrick M.; Ma, Jun; Prince, Amanda L.; Antony, Kathleen M.; Seferovic, Maxim D.; Aagaard, Kjersti M.

    2017-01-01

    Human microbial communities are characterized by their taxonomic, metagenomic, and metabolic diversity, which varies by distinct body sites and influences human physiology. However, when and how microbial communities within each body niche acquire unique taxonomical and functional signatures in early life remains underexplored. We thus sought to assess the taxonomic composition and potential metabolic function of the neonatal and early infant microbiota across multiple body sites, and assess the impact of mode of delivery and its potential confounders or modifiers. A cohort of pregnant women in their early 3rd trimester (n=81) were prospectively enrolled for longitudinal sampling through 6 weeks post-delivery, and a second matched cross-sectional cohort (n=81) was additionally recruited for sampling once at delivery. Samples were collected for each maternal-infant dyad across multiple body sites, including stool, oral gingiva, nares, skin and vagina. 16S rRNA gene sequencing analysis and whole genome shotgun sequencing was performed to interrogate the composition and function of the neonatal and maternal microbiota. We found that the neonatal microbiota and its associated functional pathways were relatively homogenous across all body sites at delivery, with the notable exception of neonatal meconium. However, by 6 weeks, the infant microbiota structure and function had significantly expanded and diversified, with body site serving as the primary determinant of the bacterial community composition and its functional capacity. Although minor variations in the neonatal (immediately at birth) microbiota community structure were associated with Cesarean delivery in some body sites (oral, nares, and skin; R2 = 0.038), this was not true in neonatal stool (meconium, Mann-Whitney p>0.05) and there was no observable difference in community function regardless of delivery mode. By 6 weeks of age, the infant microbiota structure and function had expanded and diversified with

  17. Maturation of the infant microbiome community structure and function across multiple body sites and in relation to mode of delivery.

    PubMed

    Chu, Derrick M; Ma, Jun; Prince, Amanda L; Antony, Kathleen M; Seferovic, Maxim D; Aagaard, Kjersti M

    2017-03-01

    Human microbial communities are characterized by their taxonomic, metagenomic and metabolic diversity, which varies by distinct body sites and influences human physiology. However, when and how microbial communities within each body niche acquire unique taxonomical and functional signatures in early life remains underexplored. We thus sought to determine the taxonomic composition and potential metabolic function of the neonatal and early infant microbiota across multiple body sites and assess the effect of the mode of delivery and its potential confounders or modifiers. A cohort of pregnant women in their early third trimester (n = 81) were prospectively enrolled for longitudinal sampling through 6 weeks after delivery, and a second matched cross-sectional cohort (n = 81) was additionally recruited for sampling once at the time of delivery. Samples across multiple body sites, including stool, oral gingiva, nares, skin and vagina were collected for each maternal-infant dyad. Whole-genome shotgun sequencing and sequencing analysis of the gene encoding the 16S rRNA were performed to interrogate the composition and function of the neonatal and maternal microbiota. We found that the neonatal microbiota and its associated functional pathways were relatively homogeneous across all body sites at delivery, with the notable exception of the neonatal meconium. However, by 6 weeks after delivery, the infant microbiota structure and function had substantially expanded and diversified, with the body site serving as the primary determinant of the composition of the bacterial community and its functional capacity. Although minor variations in the neonatal (immediately at birth) microbiota community structure were associated with the cesarean mode of delivery in some body sites (oral gingiva, nares and skin; R(2) = 0.038), this was not true for neonatal stool (meconium; Mann-Whitney P > 0.05), and there was no observable difference in community function regardless of delivery mode

  18. Dendrochemistry of multiple releases of chlorinated solvents at a former industrial site

    USGS Publications Warehouse

    Balouet, Jean Christophe; Burken, Joel G.; Karg, Frank; Vroblesky, Don; Smith, Kevin T.; Grudd, Hakan; Rindby, Anders; Beaujard, Francois; Chalot, Michel

    2012-01-01

    Trees can take up and assimilate contaminants from the soil, subsurface, and groundwater. Contaminants in the transpiration stream can become bound or incorporated into the annual rings formed in trees of the temperate zones. The chemical analysis of precisely dated tree rings, called dendrochemistry, can be used to interpret past plant interactions with contaminants. This investigation demonstrates that dendrochemistry can be used to generate historical scenarios of past contamination of groundwater by chlorinated solvents at a site in Verl, Germany. Increment cores from trees at the Verl site were collected and analyzed by energy-dispersive X-ray fluorescence (EDXRF) line scanning. The EDXRF profiles showed four to six time periods where tree rings had anomalously high concentrations of chlorine (Cl) as an indicator of potential contamination by chlorinated solvents.

  19. Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

    PubMed

    Payvar, F; Firestone, G L; Ross, S R; Chandler, V L; Wrange, O; Carlstedt-Duke, J; Gustafsson, J A; Yamamoto, K R

    1982-01-01

    Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

  20. Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

    SciTech Connect

    Crankshaw, D.; Gaspar, V.; Pliska, V. )

    1990-01-01

    The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.

  1. Multiple elemental exposures amongst workers at the Agbogbloshie electronic waste (e-waste) site in Ghana.

    PubMed

    Srigboh, Roland Kofi; Basu, Niladri; Stephens, Judith; Asampong, Emmanuel; Perkins, Marie; Neitzel, Richard L; Fobil, Julius

    2016-12-01

    Electronic waste (e-waste) recycling is growing worldwide and raising a number of environmental health concerns. One of the largest e-waste sites is Agbogbloshie (Ghana). While several toxic elements have been reported in Agbogbloshie's environment, there is limited knowledge of human exposures there. The objectives of this study were to characterize exposures to several essential (copper, iron, manganese, selenium, zinc) and toxic (arsenic, cadmium, cobalt, chromium, mercury, nickel, lead) elements in the urine and blood of male workers (n = 58) at Agbogbloshie, as well as females (n = 11) working in activities that serve the site, and to relate these exposures to sociodemographic and occupational characteristics. The median number of years worked at the site was 5, and the average worker indicated being active in 6.8 tasks (of 9 key e-waste job categories). Additionally, we categorized four main e-waste activities (in brackets % of population self-reported main activity): dealing (22.4%), sorting (24.1%), dismantling (50%), and burning (3.4%) e-waste materials. Many blood and urinary elements (including essential ones) were within biomonitoring reference ranges. However, blood cadmium (1.2 μg/L median) and lead (6.4 μg/dl; 67% above U.S. CDC/NIOSH reference level), and urinary arsenic (38.3 μg/L; 39% above U.S. ATSDR value) levels were elevated compared to background populations elsewhere. Workers who burned e-waste tended to have the highest biomarker levels. The findings of this study contribute to a growing body of work at Agbogbloshie (and elsewhere) to document that individuals working within e-waste sites are exposed to a number of toxic elements, some at potentially concerning levels. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Natural Attenuation of Fuel Hydrocarbons Performance and Cost Results from Multiple Air Force Demonstration Sites

    DTIC Science & Technology

    1999-10-01

    of LTM plans. One site had sufficiently complex hydrogeology to warrant the construction of a three-dimensional (3-D) numerical model using MODFLOW ...ST-C 3 14 9 285 18 77.4 Eglin AFB, FL POL Facility F-C/S, C, Peat F-C/S 0 4 6 4380 263 657 Ellsworth, SD Area D G, ST-C G 7.5 24 9 146 5 76

  3. Natural Attenuation of Chlorinated Solvents Performance and Cost Results from Multiple Air Force Demonstration Sites

    DTIC Science & Technology

    1999-10-01

    degree and rate of intrinsic bioremediation of CAHs is highly site specific, and is dependent upon the prevailing bio- and geochemistries of groundwater ...3.2 GEOCHEMISTRY AND BIODEGRADATION Contaminant biodegradation causes geochemical changes in the groundwater system as a result of native or... hydrologically diverse groundwater systems. Water Resources Research, 31:359-371. Clement, T.P. 1997. A modular computer code for simulating

  4. Dysbiosis Across Multiple Body Sites in Critically Ill Adult Surgical Patients.

    PubMed

    Yeh, Andrew; Rogers, Matthew Brian; Firek, Brian; Neal, Matthew D; Zuckerbraun, Brian S; Morowitz, Michael J

    2016-12-01

    The microbiota of critically ill patients likely undergoes dramatic changes but has not been rigorously studied with a culture-independent high-throughput approach. The aim of this study was to characterize spatial and temporal variation in the microbiota of critically ill patients. Trauma and acute surgery patients admitted to the intensive care unit (ICU) were sampled at five body sites (stool, tongue, skin, trachea, urine) every 3 to 4 days. A mean of 10.8 samples was collected from 32 patients with a mean sampling period of 8.8 days. Bacterial 16S rRNA sequences were amplified and sequenced for microbiota analyses. Results were compared to data from unhospitalized adult participants in the American Gut and Human Microbiome Projects. Relative to healthy adults, alpha diversity was decreased in ICU gut and skin samples at all time points. Diversity in tongue swabs decreased over time. Beta diversity measures indicated differences in community membership between critically ill and healthy adults at each body site. Taxonomic alterations in the ICU included depletion of important commensal bacteria such as Faecalibacterium in GI samples and Corynebacterium in skin swabs and enrichment with pathogens such as Enterococcus, Mycoplasma, and Staphylococcus. A high proportion of ICU sample sets contained pathogens present simultaneously at three body sites indicating widespread colonization. In several cases, clinically relevant airway infections were preceded by the appearance of the causative pathogen in tracheal microbiome profiles. These results demonstrate that the microbiome of critically ill patients undergoes a loss of diversity, loss of site specificity, and a shift toward dominant pathogens. These changes may provide opportunities to precisely modulate the microbiome and thereby improve patient outcomes.

  5. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    SciTech Connect

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    also contains 3-sialyllactose (another predicted site 1 binder) and bisbenzimide 33342 (non-binder). A series of five predicted Site-2 binders were then screened sequentially in the presence of the Site-1 binder doxorubicin. These experiments showed that the compounds lavendustin A and naphthofluorescein-di-({beta}-D-galactopyranoside) binds along with doxorubicin to TetC. Further experiments indicate that doxorubicin and lavendustin are potential candidates to use in preparing a bidendate inhibitor specific for TetC. The simultaneous binding of two different predicted Site-2 ligands to TetC suggests that they may bind multiple sites. Another possibility is that the conformations of the binding sites are dynamic and can bind multiple diverse ligands at a single site depending on the pre-existing conformation of the protein, especially when doxorubicin is already bound.

  6. Evolution of the Florida Launch Site Architecture: Embracing Multiple Customers, Enhancing Launch Opportunities

    NASA Technical Reports Server (NTRS)

    Colloredo, Scott; Gray, James A.

    2011-01-01

    The impending conclusion of the Space Shuttle Program and the Constellation Program cancellation unveiled in the FY2011 President's budget created a large void for human spaceflight capability and specifically launch activity from the Florida launch Site (FlS). This void created an opportunity to re-architect the launch site to be more accommodating to the future NASA heavy lift and commercial space industry. The goal is to evolve the heritage capabilities into a more affordable and flexible launch complex. This case study will discuss the FlS architecture evolution from the trade studies to select primary launch site locations for future customers, to improving infrastructure; promoting environmental remediation/compliance; improving offline processing, manufacturing, & recovery; developing range interface and control services with the US Air Force, and developing modernization efforts for the launch Pad, Vehicle Assembly Building, Mobile launcher, and supporting infrastructure. The architecture studies will steer how to best invest limited modernization funding from initiatives like the 21 st elSe and other potential funding.

  7. Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

    SciTech Connect

    Prochazkova, Katerina; Shuvalova, Ludmilla A.; Minasov, George; Voburka, Zdeněk; Anderson, Wayne F.; Satchell, Karla J.F.

    2009-10-05

    The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP{sub 6}). In this study, we demonstrated that InsP{sub 6} is not simply an allosteric cofactor, but rather binding of InsP{sub 6} stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-{angstrom} crystal structure of this InsP{sub 6}-bound unprocessed form of CPD was determined and revealed the scissile bond Leu{sup 3428}-Ala{sup 3429} captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP{sub 6}, but was reactivated for high affinity binding of InsP{sub 6} by cooperative binding of both a new substrate and InsP{sub 6}. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.

  8. Bayesian multiple-instance motif discovery with BAMBI: inference of recombinase and transcription factor binding sites

    PubMed Central

    Jajamovich, Guido H.; Wang, Xiaodong; Arkin, Adam P.; Samoilov, Michael S.

    2011-01-01

    Finding conserved motifs in genomic sequences represents one of essential bioinformatic problems. However, achieving high discovery performance without imposing substantial auxiliary constraints on possible motif features remains a key algorithmic challenge. This work describes BAMBI—a sequential Monte Carlo motif-identification algorithm, which is based on a position weight matrix model that does not require additional constraints and is able to estimate such motif properties as length, logo, number of instances and their locations solely on the basis of primary nucleotide sequence data. Furthermore, should biologically meaningful information about motif attributes be available, BAMBI takes advantage of this knowledge to further refine the discovery results. In practical applications, we show that the proposed approach can be used to find sites of such diverse DNA-binding molecules as the cAMP receptor protein (CRP) and Din-family site-specific serine recombinases. Results obtained by BAMBI in these and other settings demonstrate better statistical performance than any of the four widely-used profile-based motif discovery methods: MEME, BioProspector with BioOptimizer, SeSiMCMC and Motif Sampler as measured by the nucleotide-level correlation coefficient. Additionally, in the case of Din-family recombinase target site discovery, the BAMBI-inferred motif is found to be the only one functionally accurate from the underlying biochemical mechanism standpoint. C++ and Matlab code is available at http://www.ee.columbia.edu/~guido/BAMBI or http://genomics.lbl.gov/BAMBI/. PMID:21948794

  9. Advanced source apportionment of size-resolved trace elements at multiple sites in London during winter

    NASA Astrophysics Data System (ADS)

    Visser, S.; Slowik, J. G.; Furger, M.; Zotter, P.; Bukowiecki, N.; Canonaco, F.; Flechsig, U.; Appel, K.; Green, D. C.; Tremper, A. H.; Young, D. E.; Williams, P. I.; Allan, J. D.; Coe, H.; Williams, L. R.; Mohr, C.; Xu, L.; Ng, N. L.; Nemitz, E.; Barlow, J. F.; Halios, C. H.; Fleming, Z. L.; Baltensperger, U.; Prévôt, A. S. H.

    2015-10-01

    Trace element measurements in PM10-2.5, PM2.5-1.0 and PM1.0-0.3 aerosol were performed with 2 h time resolution at kerbside, urban background and rural sites during the ClearfLo winter 2012 campaign in London. The environment-dependent variability of emissions was characterized using the Multilinear Engine implementation of the positive matrix factorization model, conducted on data sets comprising all three sites but segregated by size. Combining the sites enabled separation of sources with high temporal covariance but significant spatial variability. Separation of sizes improved source resolution by preventing sources occurring in only a single size fraction from having too small a contribution for the model to resolve. Anchor profiles were retrieved internally by analysing data subsets, and these profiles were used in the analyses of the complete data sets of all sites for enhanced source apportionment. A total of nine different factors were resolved (notable elements in brackets): in PM10-2.5, brake wear (Cu, Zr, Sb, Ba), other traffic-related (Fe), resuspended dust (Si, Ca), sea/road salt (Cl), aged sea salt (Na, Mg) and industrial (Cr, Ni); in PM2.5-1.0, brake wear, other traffic-related, resuspended dust, sea/road salt, aged sea salt and S-rich (S); and in PM1.0-0.3, traffic-related (Fe, Cu, Zr, Sb, Ba), resuspended dust, sea/road salt, aged sea salt, reacted Cl (Cl), S-rich and solid fuel (K, Pb). Human activities enhance the kerb-to-rural concentration gradients of coarse aged sea salt, typically considered to have a natural source, by 1.7-2.2. These site-dependent concentration differences reflect the effect of local resuspension processes in London. The anthropogenically influenced factors traffic (brake wear and other traffic-related processes), dust and sea/road salt provide further kerb-to-rural concentration enhancements by direct source emissions by a factor of 3.5-12.7. The traffic and dust factors are mainly emitted in PM10-2.5 and show strong

  10. MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR

    PubMed Central

    Fushan, Alexey A; Drayna, Dennis T

    2009-01-01

    Background Multiple approaches for the site-directed mutagenesis (SDM) have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents. Results In this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA. Conclusion A wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM. PMID:19778447

  11. The Transcription Factor Bach2 Is Phosphorylated at Multiple Sites in Murine B Cells but a Single Site Prevents Its Nuclear Localization.

    PubMed

    Ando, Ryo; Shima, Hiroki; Tamahara, Toru; Sato, Yoshihiro; Watanabe-Matsui, Miki; Kato, Hiroki; Sax, Nicolas; Motohashi, Hozumi; Taguchi, Keiko; Yamamoto, Masayuki; Nio, Masaki; Maeda, Tatsuya; Ochiai, Kyoko; Muto, Akihiko; Igarashi, Kazuhiko

    2016-01-22

    The transcription factor Bach2 regulates the immune system at multiple points, including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells are still unclear. Here, we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of the B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in the mature B cell line BAL17. Among the sites identified, serine 535 (Ser-535) was critical for the regulation of Bach2 because a single mutation of Ser-535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas Ser-509 played an auxiliary role. Bach2 repressor activity was enhanced by the Ser-535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Heartburn and multiple-site foregut perforations as primary manifestation of Crohn's disease.

    PubMed

    Bona, D; Incarbone, R; Chella, B; Vecchi, M; Bonavina, L

    2005-01-01

    Crohn's disease may affect any segment of the digestive tract, more commonly the distal ileum, colon and/or perianal region. There is an increasing number of reports dealing with foregut Crohn's disease. We present the case of a patient with a history of heartburn and multiple spontaneous perforations of the esophagus, duodenum and jejunum as a primary manifestation of Crohn's disease who required emergency surgical and endoscopic procedures. Early detection of Crohn's disease may decrease the incidence of acute life-threatening complications provided that appropriate medical treatment is administered and a multidisciplinary approach is offered to these patients.

  13. Genome-Wide Assessment of Efficiency and Specificity in CRISPR/Cas9 Mediated Multiple Site Targeting in Arabidopsis

    PubMed Central

    Peterson, Brenda A.; Haak, David C.; Nishimura, Marc T.; Teixeira, Paulo J. P. L.; James, Sean R.; Dangl, Jeffery L.; Nimchuk, Zachary L.

    2016-01-01

    Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for clean mutant generation with no need to enhance specificity or adopt alternate Cas9 variants. PMID:27622539

  14. Multiple Coordination Exchanges for Room-Temperature Activation of Open-Metal Sites in Metal-Organic Frameworks.

    PubMed

    Bae, Jinhee; Choi, Jae Sun; Hwang, Sunhyun; Yun, Won Seok; Song, Dahae; Lee, JaeDong; Jeong, Nak Cheon

    2017-07-26

    The activation of open coordination sites (OCSs) in metal-organic frameworks (MOFs), i.e., the removal of solvent molecules coordinated at the OCSs, is an essential step that is required prior to the use of MOFs in potential applications such as gas chemisorption, separation, and catalysis because OCSs often serve as key sites in these applications. Recently, we developed a "chemical activation" method involving dichloromethane (DCM) treatment at room temperature, which is considered to be a promising alternative to conventional thermal activation (TA), because it does not require the application of external thermal energy, thereby preserving the structural integrity of the MOFs. However, strongly coordinating solvents such as N,N-dimethylformamide (DMF), N,N-diethylformamide (DEF), and dimethyl sulfoxide (DMSO) are difficult to remove solely with the DCM treatment. In this report, we demonstrate a multiple coordination exchange (CE) process executed initially with acetonitrile (MeCN), methanol (MeOH), or ethanol (EtOH) and subsequently with DCM to achieve the complete activation of OCSs that possess strong extracoordination. Thus, this process can serve as an effective "chemical route" to activation at room temperature that does not require applying heat. To the best of our knowledge, no previous study has demonstrated the activation of OCSs using this multiple CE process, although MeOH and/or DCM has been popularly used in pretreatment steps prior to the TA process. Using MOF-74(Ni), we demonstrate that this multiple CE process can safely activate a thermally unstable MOF without inflicting structural damage. Furthermore, on the basis of in situ (1)H nuclear magnetic resonance ((1)H NMR) and Raman studies, we propose a plausible mechanism for the activation behavior of multiple CE.

  15. Multiple-site and-year analyses of stream insect emergence: a test of ecological theory.

    PubMed

    Statzner, Bernhard; Resh, Vincent H

    1993-10-01

    The use of existing data sets to test applicability of existing ecological theory is an uncommon but potentially cost-effective approach for exploitation of previously accumulated knowledge. Studies on the emergence of insects from small streams have been a major research topic in aquatic ecology, particularly in Austria and Germany; the availability of emergence data from these two countries, covering over 1 million identified specimens, from 18 sites, and for 32 collection years is an example of such exploitable information. Concurrent estimates of annual emergence biomass and annual benthic secondary production for 18 aquatic insect populations showed a statistically significant relationship, contradicting the premise that emergence data do not provide any quantitative measure for a given stream area. Therefore, the emergence data were examined to test various predictions from ecological theory. Observed richness of emerging species of three orders of lotic insects - the Ephemeroptera, Plecoptera, and Trichoptera (EPT) - over 15 years at one site did not agree with predictions based on either flow predictability or change in flow and the "habitat templet concept". Trends in observed richness of emerging EPT species over 1 year at 18 sites agreed weakly with predictions using either pH values or the annual temperature amplitude and the "intermediate disturbance hypothesis", or using either annual temperature amplitude or total biomass of EPT emergence and the "disturbance-productivity-diversity model". A prediction of the "river continuum concept" that abundance of the shredder functional-feeding group should decrease and that of grazers should increase along a dense- to open-canopy gradient was not consistently supported by the emergence biomass data. For shredders and grazers of all insects (12 sites) and EPT (18 sites), this trend was apparent (but not significant) only if sites with intermediate canopy density were omitted. We identified three critical

  16. HPV Prevalence in Multiple Anatomical Sites among Men Who Have Sex with Men in Peru.

    PubMed

    Blas, Magaly M; Brown, Brandon; Menacho, Luis; Alva, Isaac E; Silva-Santisteban, Alfonso; Carcamo, Cesar

    2015-01-01

    Human Papilloma Virus (HPV) infection is the most common sexually transmitted viral infection worldwide. HPV is highly prevalent in sexually active men who have sex with men (MSM) and has been associated with anal cancer, penile cancer, and oropharyngeal cancer. From March to September 2011, we conducted a cross-sectional study of HPV prevalence among MSM above age 18 years. Participants were recruited using respondent driven sampling at Clinica Cayetano Heredia. All participants provided anal, genital, and oral samples for HPV DNA testing, and blood for HIV and HPV antibody testing. A total of 200 MSM were recruited in the study. The mean age was 34 years (range 18-59 years, SD = 9.4) and101 participants were HIV negative (99 HIV positive). HPV 6/11/16/18 or quadrivalent HPV vaccine (HPV4) genotype seroprevalence among HIV negative and positive MSM was 64.3% (55%-75.9%) and 93.8% (87.6%-99.2%) respectively (p<0.001). HIV positivity was associated with a higher prevalence of HPV4 and HPV 16/18 DNA at external genital sites and the anal canal. HPV4 DNA prevalence at external genital sites among HIV negative and positive MSM was 14.9% and 28.7% (p = 0.02) respectively, at anal canal was 50.9% and 79.0% (p = 0.001), and at the oral cavity was 9.9% and 8.5% (p = 0.6). HPV4 seroprevalence was high in our study among both HIV positives and negatives, with HPV DNA prevalence much lower, and the anal canal being the anatomical site with the highest HPV DNA prevalence. HPV prevention interventions are needed among MSM at high-risk for HIV infection.

  17. HPV Prevalence in Multiple Anatomical Sites among Men Who Have Sex with Men in Peru

    PubMed Central

    Blas, Magaly M.; Brown, Brandon; Menacho, Luis; Alva, Isaac E.; Silva-Santisteban, Alfonso; Carcamo, Cesar

    2015-01-01

    Background Human Papilloma Virus (HPV) infection is the most common sexually transmitted viral infection worldwide. HPV is highly prevalent in sexually active men who have sex with men (MSM) and has been associated with anal cancer, penile cancer, and oropharyngeal cancer. Methods From March to September 2011, we conducted a cross-sectional study of HPV prevalence among MSM above age 18 years. Participants were recruited using respondent driven sampling at Clinica Cayetano Heredia. All participants provided anal, genital, and oral samples for HPV DNA testing, and blood for HIV and HPV antibody testing. Results A total of 200 MSM were recruited in the study. The mean age was 34 years (range 18–59 years, SD = 9.4) and101 participants were HIV negative (99 HIV positive). HPV 6/11/16/18 or quadrivalent HPV vaccine (HPV4) genotype seroprevalence among HIV negative and positive MSM was 64.3% (55%-75.9%) and 93.8% (87.6%-99.2%) respectively (p<0.001). HIV positivity was associated with a higher prevalence of HPV4 and HPV 16/18 DNA at external genital sites and the anal canal. HPV4 DNA prevalence at external genital sites among HIV negative and positive MSM was 14.9% and 28.7% (p = 0.02) respectively, at anal canal was 50.9% and 79.0% (p = 0.001), and at the oral cavity was 9.9% and 8.5% (p = 0.6). Conclusions HPV4 seroprevalence was high in our study among both HIV positives and negatives, with HPV DNA prevalence much lower, and the anal canal being the anatomical site with the highest HPV DNA prevalence. HPV prevention interventions are needed among MSM at high-risk for HIV infection. PMID:26437318

  18. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

  19. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  20. Complex magnetic order from multiple Ce-sites in Ce3Pd4Sn6

    NASA Astrophysics Data System (ADS)

    Scheidt, E.-W.; Fischer, A.; Wybranski, O.; Eickerling, G.; Scherer, W.; Baenitz, M.; Mayr, F.; Heying, B.; Pöttgen, R.

    2017-08-01

    Polycrystalline \\text{C}{{\\text{e}}3} \\text{P}{{\\text{d}}4} \\text{S}{{\\text{n}}6} samples were synthesized by arc-melting and subsequent annealing at 970 K. Specific heat, electrical resistivity and magnetic susceptibility measurements are performed over a wide range in temperature and provide hints for the presence of a complex magnetic ordering below 3 K arising from three crystallographically independent Ce sites. This behaviour is driven by a complex interplay between ferro-, ferri-, and antiferromagnetic correlations among the Ce atoms.

  1. Communication: Active space decomposition with multiple sites: density matrix renormalization group algorithm.

    PubMed

    Parker, Shane M; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE(h) or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  2. Multiple ligand simultaneous docking: orchestrated dancing of ligands in binding sites of protein.

    PubMed

    Li, Huameng; Li, Chenglong

    2010-07-30

    Present docking methodologies simulate only one single ligand at a time during docking process. In reality, the molecular recognition process always involves multiple molecular species. Typical protein-ligand interactions are, for example, substrate and cofactor in catalytic cycle; metal ion coordination together with ligand(s); and ligand binding with water molecules. To simulate the real molecular binding processes, we propose a novel multiple ligand simultaneous docking (MLSD) strategy, which can deal with all the above processes, vastly improving docking sampling and binding free energy scoring. The work also compares two search strategies: Lamarckian genetic algorithm and particle swarm optimization, which have respective advantages depending on the specific systems. The methodology proves robust through systematic testing against several diverse model systems: E. coli purine nucleoside phosphorylase (PNP) complex with two substrates, SHP2NSH2 complex with two peptides and Bcl-xL complex with ABT-737 fragments. In all cases, the final correct docking poses and relative binding free energies were obtained. In PNP case, the simulations also capture the binding intermediates and reveal the binding dynamics during the recognition processes, which are consistent with the proposed enzymatic mechanism. In the other two cases, conventional single-ligand docking fails due to energetic and dynamic coupling among ligands, whereas MLSD results in the correct binding modes. These three cases also represent potential applications in the areas of exploring enzymatic mechanism, interpreting noisy X-ray crystallographic maps, and aiding fragment-based drug design, respectively.

  3. Multiplicity

    DTIC Science & Technology

    1991-04-01

    practice as a "[descent] into that inner circle of the Inferno where the damned endlessly degate multiplicity for sentencing." United States v. Barnard...select the charges to be brought in a particular case"). 19 Brown v. Ohio, 432 U.S. 161, 165 (1977). 20 Whalen v. United States, 445 U.S. at 689. 21...parte Lange, 8-5 U.S. (19 Wall.) 163 (1874). Cf. Brown v. Ohio, 432 U.S. at 165 ("once the legislature has acted courts may not impose more than one

  4. Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.

    PubMed

    Young, Isaac A; Mittal, Chitvan; Shogren-Knaak, Michael A

    2016-02-01

    Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.

  5. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III.

    PubMed

    Taddese, Samuel; Jung, Michael C; Ihling, Christian; Heinz, Andrea; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III. Copyright 2009 Elsevier B.V. All rights reserved.

  6. Aminopyridinyl-Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites.

    PubMed

    Okamura, Hidenori; Taniguchi, Yosuke; Sasaki, Shigeki

    2016-09-26

    The sequence-specific formation of triplex DNA offers a potential basis for genome-targeting technologies. In an antiparallel triplex DNA, the sequence-specificity is established by the formation of specific base triplets (G-GC, A-AT, and T-AT) between a triplex-forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long-standing goal for the triplex-forming technology. We now describe the design and synthesis of pseudo-deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex-forming sequence. The aminopyridine-bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3-methyl-2-aminopyridinyl-ΨdC ((Me) AP-ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, (Me) AP-ΨdC is expected to serve as a new starting point of triplex-forming oligonucleotides for a wide variety of genome-targeting applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    SciTech Connect

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2008-01-01

    Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal a-amino group and the -amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal a-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.

  8. Partitioning of ferrocenium ions between multiple redox sites on spinacch plastocyanin

    SciTech Connect

    Pladziewicz, J.R.; Brenner, M.S.

    1987-10-21

    Kinetic measurements of the oxidation of plastocyanin (spinach) by ferrocenium, (hydroxymethyl)ferrocenium, and (chloromercurio)ferrocenium in the presence of the redox-inactive inhibitors ((NH/sub 3/)/sub 5/CoNH/sub 2/Co(NH/sub 3/)/sub 5/)/sup 5 +/, (Co(NH/sub 3/)/sub 6/)/sup 3 +/, and (Zr-(C/sub 2/O/sub 4/)/sub 4/)/sup 4 -/ as a function of pH at 25/sup 0/C and ..mu.. = 0.10 are reported. These results support a mechanism in which electrons are transferred from two locations on plastocyanin's surface; one site being the hydrophobic pocket near His-87 where copper is closest to the surface of the protein; the second being the hydrophilic, negatively charged region surrounding Tyr-83. The oxidizing agent's charge and ligand hydrophobicity and the solution pH are found to control the partitioning of the oxidizing agent between the two active sites, and the relative importance of oxidizing agent charge and hydrophobicity in this partitioning is assessed. Electron transfer from reduced plastocyanin is found to be markedly dependent on pH (pK/sub a/ = 5.03 +- 0.06), with the protonated protein being redox inactive.

  9. Polarized light spectroscopy for measurement of the microvascular response to local heating at multiple skin sites.

    PubMed

    Tesselaar, Erik; Bergkvist, Max; Sjöberg, Folke; Farnebo, Simon

    2012-11-01

    To evaluate whether TiVi, a technique based on polarized light, could measure the change in RBC concentration during local heating in healthy volunteers. Using a custom-made transparent heater, forearm skin was heated to 42 °C for 40 minutes while the change in RBC concentration was measured with TiVi. The perfusion response during local heating was measured at the same time with Laser Doppler flowmetry. Mean RBC concentration increased (91 ± 34 vs. 51 ± 34 A.U. at baseline, p < 0.001). The spatial heterogeneity of the RBC concentration in the measured skin areas was 26 ± 6.4% at baseline, and 23 ± 4.6% after 40 minutes of heating. The mean RBC concentrations in two skin sites were highly correlated (0.98 at baseline and 0.96 after 40 minutes of heating). The change in RBC concentration was less than the change in perfusion, measured with LDF. Unlike with LDF, a neurally mediated peak was not observed with TiVi in most of the test subjects. TiVi is a valuable technique for measuring the microvascular response to local heating in the skin, and offers a high reproducibility for simultaneous measurements at different skin sites, provided carefully controlled experiments are ensured. © 2012 John Wiley & Sons Ltd.

  10. Prediction of phosphorylation sites based on the integration of multiple classifiers.

    PubMed

    Han, R Z; Wang, D; Chen, Y H; Dong, L K; Fan, Y L

    2017-02-23

    Phosphorylation is an important part of post-translational modifications of proteins, and is essential for many biological activities. Phosphorylation and dephosphorylation can regulate signal transduction, gene expression, and cell cycle regulation in many cellular processes. Phosphorylation is extremely important for both basic research and drug discovery to rapidly and correctly identify the attributes of a new protein phosphorylation sites. Moreover, abnormal phosphorylation can be used as a key medical feature related to a disease in some cases. The using of computational methods could improve the accuracy of detection of phosphorylation sites, which can provide predictive guidance for the prevention of the occurrence and/or the best course of treatment for certain diseases. Furthermore, this approach can effectively reduce the costs of biological experiments. In this study, a flexible neural tree (FNT), particle swarm optimization, and support vector machine algorithms were used to classify data with secondary encoding according to the physical and chemical properties of amino acids for feature extraction. Comparison of the classification results obtained from the three classifiers showed that the classification of the FNT was the best. The three classifiers were then integrated in the form of a minority subordinate to the majority vote to obtain the results. The performance of the integrated model showed improvement in sensitivity (87.41%), specificity (87.60%), and accuracy (87.50%).

  11. Signatures of complex magnetic topologies from multiple reconnection sites induced by Kelvin-Helmholtz instability

    NASA Astrophysics Data System (ADS)

    Vernisse, Y.; Lavraud, B.; Eriksson, S.; Gershman, D. J.; Dorelli, J.; Pollock, C.; Giles, B.; Aunai, N.; Avanov, L.; Burch, J.; Chandler, M.; Coffey, V.; Dargent, J.; Ergun, R. E.; Farrugia, C. J.; Génot, V.; Graham, D. B.; Hasegawa, H.; Jacquey, C.; Kacem, I.; Khotyaintsev, Y.; Li, W.; Magnes, W.; Marchaudon, A.; Moore, T.; Paterson, W.; Penou, E.; Phan, T. D.; Retino, A.; Russell, C. T.; Saito, Y.; Sauvaud, J.-A.; Torbert, R.; Wilder, F. D.; Yokota, S.

    2016-10-01

    The Magnetospheric Multiscale mission has demonstrated the frequent presence of reconnection exhausts at thin current sheets within Kelvin-Helmholtz (KH) waves at the flank magnetopause. Motivated by these recent observations, we performed a statistical analysis of the boundary layers on the magnetosheath side of all KH current sheets on 8 September 2015. We show 86% consistency between the exhaust flows and particle leakage in the magnetosheath boundary layers but further highlight the very frequent presence of additional boundary layer signatures that do not come from the locally observed reconnection exhausts. These additional electron and ion boundary layers, of various durations and at various positions with respect to the leading and trailing boundaries of the KH waves, signal connections to reconnection sites at other locations. Based on the directionality and extent of these layers, we provide an interpretation whereby complex magnetic topologies can arise within KH waves from the combination of reconnection in the equatorial plane and at midlatitudes in the Southern and Northern Hemispheres, where additional reconnection sites are expected to be triggered by the three-dimensional field lines interweaving induced by the KH waves at the flanks (owing to differential flow and magnetic field shear with latitude). The present event demonstrates that the three-dimensional development of KH waves can induce plasma entry (through reconnection at both midlatitude and equatorial regions) already sunward of the terminator where the instability remains in its linear stage.

  12. Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

    PubMed

    Zhuo, L; Reed, K M; Phillips, R B

    1995-06-01

    Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

  13. Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion

    PubMed Central

    McLean, Meghan M; Chang, Yong; Dhar, Gautam; Heiss, John K; Johnson, Reid C

    2013-01-01

    Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. DOI: http://dx.doi.org/10.7554/eLife.01211.001 PMID:24151546

  14. The impact of multiple splice sites in human L1 elements

    PubMed Central

    Belancio, V. P.; Roy-Engel, A. M.; Deininger, P.

    2008-01-01

    LINE-1 elements represent a significant proportion of mammalian genomes. The impact of their activity on the structure and function of the host genomes has been recognized from the time of their discovery as an endogenous source of insertional mutagenesis. L1 elements contain numerous functional internal polyadenylation signals and splice sites that generate a variety of processed L1 transcripts. These sites are also reported to contribute to the generation of hybrid transcripts between L1 elements and host genes. Using northern blot analysis we demonstrate that L1 splicing, but not L1 polyadenylation, is delayed during the course of L1 expression. L1 splicing can also be negatively regulated by EBV SM protein known to alter mRNA splicing. These results suggest a potential for L1 mRNA processing to be regulated in a tissue- and/or development-specific manner. The delay in L1 splicing may also serve to protect host genes from the excessive burden of L1 interference with their normal expression via aberrant splicing. PMID:18261861

  15. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    PubMed Central

    Demirci, Hasan; Gregory, Steven T.; Dahlberg, Albert E.; Jogl, Gerwald

    2008-01-01

    SUMMARY Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal α-amino group and the ε-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal α-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA. PMID:18611379

  16. The gingival vein as a minimally traumatic site for multiple blood sampling in guinea pigs and hamsters.

    PubMed

    Rodrigues, Mariana Valotta; de Castro, Simone Oliveira; de Albuquerque, Cynthia Zaccanini; Mattaraia, Vânia Gomes de Moura; Santoro, Marcelo Larami

    2017-01-01

    Laboratory animals are still necessary in scientific investigation and vaccine testing, but while novel methodological approaches are not available for their replacement, the search for new, humane, easy, and painless methods is necessary to diminish their stress and pain. When multiple blood samples are to be collected from hamsters and guinea pigs, the number of available venipuncture sites-which are greatly diminished in these species in comparison with other rodents due to the absence of a long tail-, harasses animal caregivers and researchers. Thus, this study aimed to evaluate if gingival vein puncture could be used as an additional route to obtain multiple blood samples from anesthetized hamsters and guinea pigs in such a way that animal behavior, well-being or hematological parameters would not be altered. Thus, twelve anesthetized Syrian golden hamsters and English guinea pigs were randomly allocated in two groups: a control group, whose blood samples were not collected, and an experimental group in which blood samples (200 microliters) were collected by gingival vein puncture at weekly intervals over six weeks. Clinical assessment, body weight gain and complete blood cell count were evaluated weekly, and control and experimental animals were euthanized at week seven, when the mentolabial region was processed to histological analyses. Multiple blood sampling from the gingival vein evoked no clinical manifestations or alteration in the behavioral repertoire, nor a statistically significant difference in weight gain in both species. Guinea pigs showed no alteration in red blood cell, leukocyte or platelet parameters over time. Hamsters developed a characteristic pattern of age-related physiological changes, which were considered normal. Histological analyses showed no difference in morphological structures in the interdental gingiva, confirming that multiple blood sampling is barely traumatic. Thus, these results evidence that blood collection from multiple

  17. HPLC-purified 2-(/sup 125/I)iodomelatonin labels multiple binding sites in hamster brain

    SciTech Connect

    Niles, L.P.; Pickering, D.S.; Sayer, B.G.

    1987-09-30

    Binding of 2-(/sup 125/I)iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.

  18. A challenging diagnosis: case report of extensive pyoderma gangrenosum at multiple sites

    PubMed Central

    Ye, Mingwei J; Ye, Joshua Mingsheng; Wu, Leonard; Keating, Cameron P; Choi, Wai-Ting

    2014-01-01

    Background Pyoderma gangrenosum (PG) is a rare dermatological condition characterized by the rapid progression of a painful, necrolytic ulcer with an irregular, undermined border and commonly affects the lower extremities, mainly in the pretibial area. The diagnosis of PG is not easy. Due to lack of diagnostic laboratory test and histopathological findings indicative of PG, it is often misdiagnosed as an infection. This results in delayed or inappropriate treatment of the condition, which leads to devastating consequences such as limb amputation and death. Main observations We report a rare case of a 51-year-old female who was initially diagnosed as having infected ulcers and underwent serial debridements, which resulted in extensive PG at three different sites (abdominal, left thigh, and sacral). Conclusion This case highlights the challenges in diagnosing PG, emphasizes the key clinical features to aid diagnosis, and the clinical consequences of delayed or misdiagnosis of this condition. PMID:24741322

  19. A Compact Microelectrode Array Chip with Multiple Measuring Sites for Electrochemical Applications

    PubMed Central

    Dimaki, Maria; Vergani, Marco; Heiskanen, Arto; Kwasny, Dorota; Sasso, Luigi; Carminati, Marco; Gerrard, Juliet A.; Emneus, Jenny; Svendsen, Winnie E.

    2014-01-01

    In this paper we demonstrate the fabrication and electrochemical characterization of a microchip with 12 identical but individually addressable electrochemical measuring sites, each consisting of a set of interdigitated electrodes acting as a working electrode as well as two circular electrodes functioning as a counter and reference electrode in close proximity. The electrodes are made of gold on a silicon oxide substrate and are passivated by a silicon nitride membrane. A method for avoiding the creation of high edges at the electrodes (known as lift-off ears) is presented. The microchip design is highly symmetric to accommodate easy electronic integration and provides space for microfluidic inlets and outlets for integrated custom-made microfluidic systems on top. PMID:24878592

  20. A compact microelectrode array chip with multiple measuring sites for electrochemical applications.

    PubMed

    Dimaki, Maria; Vergani, Marco; Heiskanen, Arto; Kwasny, Dorota; Sasso, Luigi; Carminati, Marco; Gerrard, Juliet A; Emneus, Jenny; Svendsen, Winnie E

    2014-05-28

    In this paper we demonstrate the fabrication and electrochemical characterization of a microchip with 12 identical but individually addressable electrochemical measuring sites, each consisting of a set of interdigitated electrodes acting as a working electrode as well as two circular electrodes functioning as a counter and reference electrode in close proximity. The electrodes are made of gold on a silicon oxide substrate and are passivated by a silicon nitride membrane. A method for avoiding the creation of high edges at the electrodes (known as lift-off ears) is presented. The microchip design is highly symmetric to accommodate easy electronic integration and provides space for microfluidic inlets and outlets for integrated custom-made microfluidic systems on top.

  1. Modelling equilibrium shoreline response: application across multiple sites and minimum data collection requirements

    NASA Astrophysics Data System (ADS)

    Splinter, K.; Davidson, M. A.; Turner, I. L.

    2012-12-01

    The ability to predict shoreline variability and trends for a range of potential future climate scenarios is of increasing interest to coastal scientists. Here we introduce a new shoreline equilibrium model driven by cross-shore processes via changes in non-dimensional fall velocity (i.e., sediment characteristics and wave steepness) and offshore wave power (Davidson et al., submitted). The equilibrium shoreline position is modeled based on a weighted time-average of past non-dimensional fall velocity following the work of Wright et al. (1985). When the prevailing wave conditions are steeper than the time varying equilibrium, the shoreline erodes as sand is expected to move offshore and form a breaker bar. Conversely, when waves are flatter, sand moves onshore and the shoreline builds seawards. The model is applied at a number of sites within Australia and the US to quantify model skill, and to examine free parameter sensitivity and generic transferability between differing sites. Further testing using real-world and synthetic data sets is used to determine the minimum data requirements for calibration of the model, providing useful guidance to future coastline monitoring program requirements. Model hindcasts at six locations within Australia indicate this simple cross-shore equilibrium shoreline model is capable of reproducing multi-year shoreline variability with significant skill. Shoreline variance explained by the model (currently driven only by cross-shore processes) is between 40% (downdrift of an Artificial surfing reef on an exposed open coast) to 66% percent (on a central location of a storm-driven embayed beach, Fig. 1).Hindcast results for the Narrabeen embayment at alongshore locations y=2200m (a), 2600m (b) and 3200m (c). R 2 ranged from 0.57 (y=2600m) to 0.66 (y=3200m). The thickness of the data curve (grey) indicates the potential measurement error.

  2. Determination of anesthetic molecule environments by infrared spectroscopy. II. Multiple sites for nitrous oxide in proteins, lipids, and brain tissue.

    PubMed

    Hazzard, J H; Gorga, J C; Caughey, W S

    1985-08-01

    The presence of molecules of the general anesthetic nitrous oxide (N2O) in oils, esters, proteins, red cells, cream, lipid vesicles, and brain tissue upon exposure to the gas was observed by infrared spectroscopy. Analysis of the N-N-O antisymmetric stretch band reveals a distribution of N2O molecules among several sites of differing polarity in these solutions and tissues. The sensitivity of the band intensity and frequency to the number and strength of the dipoles in the solvating molecules is demonstrated by the resolution of N2O-ester and N2O-alkane interactions in acetic acid ethyl ester and oleic acid methyl ester. In all aqueous solutions and in all tissues a population of N2O molecules in water is observed. At least two sites of N2O-protein interaction are observed in purified hemoglobin A and packed red cells; multiple N2O sites may also be present in bovine serum albumin. Two sites of N2O-lipid interaction are observed in whipping cream and in an aqueous suspension of phosphatidylcholine vesicles. The sites providing the least polar immediate environment to N2O in hemoglobin, cream, and vesicles give similar band frequencies to those found in pure alkane solvents. Infrared spectra of bovine brain tissue, upon exposure to N2O, show N2O molecules present in water and in two less-polar environments. Analysis of spectra of N2O in cerebellum tissue removed from a dog under halothane-N2O anesthesia reveals, in addition to N2O in water, a single population of N2O molecules in an alkane-like environment. Infrared spectroscopy provides a unique means of probing the structure of the environment of N2O and should prove useful in correlating anesthetic potency with anesthetic environment under physiological conditions.

  3. Uncertainty Analysis on an Operational Simplified Surface Energy Balance algorithm for Estimation of Evapotranspiration at Multiple Flux Tower Sites

    NASA Astrophysics Data System (ADS)

    Chen, M.; Senay, G. B.; Verdin, J. P.; Rowland, J.

    2014-12-01

    Current regional to global and daily to annual Evapotranspiration ( ET) estimation mainly relies on surface energy balance (SEB) ET models or statistical empirical methods driven by remote sensing data and various meteorology databases. However, these ET models face challenging issues—large uncertainties from inevitable input errors, poorly defined parameters, and inadequate model structures. The eddy covariance measurements on water, energy, and carbon fluxes at globally available FLUXNET tower sites provide a feasible opportunity to assess the ET modelling uncertainties. In this study, we focused on uncertainty analysis on an operational simplified surface energy balance (SSEBop) algorithm for ET estimation at multiple Ameriflux tower sites with diverse land cover characteristics and climatic conditions. The input land surface temperature (LST) data of the algorithm were adopted from the 8-day composite1-km Moderate Resolution Imaging Spectroradiometer (MODIS) land surface temperature. The other input data were taken from the Ameriflux database. Results of statistical analysis indicated that uncertainties or random errors from input variables and parameters of SSEBop led to daily and seasonal ET estimates with relative errors around 20% across multiple flux tower sites distributed across different biomes. This uncertainty of SSEBop lies in the error range of 20-30% of similar SEB-based ET algorithms, such as, Surface Energy Balance System and Surface Energy Balance Algorithm for Land. The R2 between daily and seasonal ET estimates by SSEBop and ET eddy covariance measurements at multiple Ameriflux tower sites exceed 0.7, and even up to 0.9 for croplands, grasslands, and forests, suggesting systematic error or bias of the SSEBop is acceptable. In summary, the uncertainty assessment verifies that the SSEBop is a reliable method for wide-area ET calculation and especially useful for detecting drought years and relative drought severity for agricultural production

  4. Comparison of canine core bone marrow biopsies from multiple sites using different techniques and needles.

    PubMed

    Abrams-Ogg, Anthony C G; Defarges, Alice; Foster, Robert A; Bienzle, Dorothee

    2012-06-01

    Commonly used 11 or 13ga needles are relatively large for collection of bone marrow (BM) biopsies from small dogs. The aims of this study were to assess ease of BM collection and quality of specimens obtained from small dogs using 13 and 15ga needles and to determine if specimen encasement would improve quality. Humeral and iliac biopsies obtained from 17 Beagle dogs with a 15ga needle and a power driver were compared with humeral biopsies obtained with a 13ga Jamshidi needle. Ease of collection (scored as 1 [very difficult] to 5 [very easy] for 3 components of collection with scores summed), section quality (scored as 1 [poor] to 5 [good]), and lesions at collection sites were scored. Quality of additional humeral biopsies obtained with a 15ga needle and wrapped in tissue paper prior to fixation was assessed. Use of a 15ga needle to obtain a humeral BM biopsy was significantly easier (mean score ± SD = 13.6 ± 1.7) than obtaining humeral BM using a 13ga needle (11.4 ± 1.6; P < .001) or obtaining iliac BM using a 15ga needle (10.9 ± 2.0, P < .001). Quality of humeral biopsies obtained with a 13ga needle (3.9 ± 1.2) was better than for biopsies of the humerus (1.9 ± 1.3, P < .001) or ilium (1.4 ± 0.6, P < .001) using a 15ga needle. Only sites sampled with a 13ga needle were identifiable grossly after the procedure. In most biopsies, cell density and cellularity were lower when a 15ga needle was used. Paper-wrapping of biopsies did not improve quality. In small dogs, collection of humeral BM biopsies using a 15g needle is feasible and more easily accomplished than collection using a 13ga needle. Hematopoiesis may be underestimated in specimens collected using the smaller needle. © 2012 American Society for Veterinary Clinical Pathology.

  5. A regional flux-based risk assessment approach for multiple contaminated sites on groundwater bodies.

    PubMed

    Jamin, P; Dollé, F; Chisala, B; Orban, Ph; Popescu, I-C; Hérivaux, C; Dassargues, A; Brouyère, S

    2012-01-01

    In the context of the Water Framework Directive (EP and CEU, 2000), management plans have to be set up to monitor and to maintain water quality in groundwater bodies in the EU. In heavily industrialized and urbanized areas, the cumulative effect of multiple contaminant sources is likely and has to be evaluated. In order to propose adequate measures, the calculated risk should be based on criteria reflecting the risk of groundwater quality deterioration, in a cumulative manner and at the scale of the entire groundwater body. An integrated GIS- and flux-based risk assessment approach for groundwater bodies is described, with a regional scale indicator for evaluating the quality status of the groundwater body. It is based on the SEQ-ESO currently used in the Walloon Region of Belgium which defines, for different water uses and for a detailed list of groundwater contaminants, a set of threshold values reflecting the levels of water quality and degradation with respect to each contaminant. The methodology is illustrated with first results at a regional scale on a groundwater body-scale application to a contaminated alluvial aquifer which has been classified to be at risk of not reaching a good quality status by 2015. These first results show that contaminants resulting from old industrial activities in that area are likely to contribute significantly to the degradation of groundwater quality. However, further investigations are required on the evaluation of the actual polluting pressures before any definitive conclusion be established.

  6. Bayesian estimation of inflow hydrographs in ungauged sites of multiple reach systems

    NASA Astrophysics Data System (ADS)

    D'Oria, Marco; Mignosa, Paolo; Tanda, Maria Giovanna

    2014-01-01

    A Bayesian Geostatistical Approach to evaluate unknown upstream flow hydrographs in multiple reach systems is implemented. The methodology was, firstly, tested through three synthetic examples of river confluences, that differ in the available data, boundary conditions and number of the estimated inflow time series. Input discharge hydrographs were routed downstream by means of the widely known HEC-RAS river analysis system to obtain the downstream stage hydrographs used as known observations for the reverse procedure. In almost all cases, the observed water levels were corrupted with random errors to highlight the reliability of the methodology in preventing instabilities and overfitting. Then the procedure was applied to the real case study of the Parma-Baganza river confluence located at the city of Parma (Italy) to assess the tributary Baganza River inflow hydrograph (supposed completely ungauged) using water level data collected downstream on the main reach. The results show that the methodology properly reproduces the unknown inflows even in presence of errors affecting the downstream water levels. The practical applicability of the proposed approach is also demonstrated in complex river systems.

  7. Infrared multiple photon dissociation spectroscopy of ciprofloxacin: Investigation of the protonation site

    NASA Astrophysics Data System (ADS)

    Bodo, E.; Ciavardini, A.; Giardini, A.; Paladini, A.; Piccirillo, S.; Rondino, F.; Scuderi, D.

    2012-04-01

    The vibrational spectrum of isolated protonated ciprofloxacin was recorded in the range 1100-2000 cm-1 by means of infrared multiple photon dissociation (IRMPD) spectroscopy. The spectrum was obtained by electrospraying a methanol solution of ciprofloxacin in a Paul ion trap, coupled to the tunable IR radiation of a free electron laser. This spectroscopic study has been complemented by quantum chemical calculations at the DFT and MP2 levels of theory to identify the possible structures present under our experimental conditions. Several low-energy isomers with protonation occurring at the piperazinyl amino group and at the carbonyl group are predicted in the energy range 0-84 kJ mol-1. A good agreement between the measured IRMPD spectrum and the calculated absorption spectrum is observed for the isomer protonated at the piperazinyl amino group. This isomer is calculated at MP2 level of theory to lie about 76 kJ/mol above the most stable isomer which is protonated at the quinone carbonyl group. This discrepancy can be rationalized by assuming that the protonation at the piperazinyl amino group, typical of the zwitterionic form that is found in protic solvents, is retained in the ESI process. The vibrational bands observed in the IRMPD spectrum are assigned to normal modes of the isomer protonated at the piperazinyl amino group, with deviations of less than 20 cm-1 between measured and calculated frequencies.

  8. DataSHIELD: an ethically robust solution to multiple-site individual-level data analysis.

    PubMed

    Budin-Ljøsne, Isabelle; Burton, Paul; Isaeva, Julia; Gaye, Amadou; Turner, Andrew; Murtagh, Madeleine J; Wallace, Susan; Ferretti, Vincent; Harris, Jennifer R

    2015-01-01

    DataSHIELD (Data Aggregation Through Anonymous Summary-statistics from Harmonised Individual levEL Databases) has been proposed to facilitate the co-analysis of individual-level data from multiple studies without physically sharing the data. In a previous paper, we investigated whether DataSHIELD could protect participant confidentiality in accordance with UK law. In this follow-up paper, we investigate whether DataSHIELD addresses a broader range of ethics-related data-sharing concerns. Ethics-related data-sharing concerns of Institutional Review Boards, ethics experts, international research consortia and research participants were identified through a literature search and systematically examined at a multidisciplinary workshop to determine whether DataSHIELD proposes mechanisms which can address these concerns. DataSHIELD addresses several ethics-related data-sharing concerns related to privacy, confidentiality, and the protection of the research participant's rights while sharing data and after the data have been shared. The data remain entirely under the direct management of the study that collected them. Data processing commands are strictly supervised, and the data are queried in a protected environment. Issues related to the return of individual research results when data are shared are eliminated; the responsibility for return remains at the study of origin. DataSHIELD can provide an innovative and robust solution for addressing commonly encountered ethics-related data-sharing concerns. © 2014 S. Karger AG, Basel.

  9. Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

    PubMed

    Tundo, Grazia R; Di Muzio, Elena; Ciaccio, Chiara; Sbardella, Diego; Di Pierro, Donato; Polticelli, Fabio; Coletta, Massimo; Marini, Stefano

    2016-10-01

    Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ)1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium.

  10. Does wine glass size influence sales for on-site consumption? A multiple treatment reversal design.

    PubMed

    Pechey, Rachel; Couturier, Dominique-Laurent; Hollands, Gareth J; Mantzari, Eleni; Munafò, Marcus R; Marteau, Theresa M

    2016-06-07

    Wine glass size can influence both perceptions of portion size and the amount poured, but its impact upon purchasing and consumption is unknown. This study aimed to examine the impact of wine glass size on wine sales for on-site consumption, keeping portion size constant. In one establishment (with separate bar and restaurant areas) in Cambridge, England, wine glass size (Standard; Larger; Smaller) was changed over eight fortnightly periods. The bar and restaurant differ in wine sales by the glass vs. by the bottle (93 % vs. 63 % by the glass respectively). Daily wine volume purchased was 9.4 % (95 % CI: 1.9, 17.5) higher when sold in larger compared to standard-sized glasses. This effect seemed principally driven by sales in the bar area (bar: 14.4 % [3.3, 26.7]; restaurant: 8.2 % [-2.5, 20.1]). Findings were inconclusive as to whether sales were different with smaller vs. standard-sized glasses. The size of glasses in which wine is sold, keeping the portion size constant, can affect consumption, with larger glasses increasing consumption. The hypothesised mechanisms for these differential effects need to be tested in a replication study. If replicated, policy implications could include considering glass size amongst alcohol licensing requirements. ISRCTN registry: ISRCTN12018175 . Registered 12(th) May 2015.

  11. Testing congruence among multiple grazing indicators: a multi-site study across the Tibetan plateau

    NASA Astrophysics Data System (ADS)

    Wang, Yun; Lehnert, Lukas; Holzapfel, Maika; Schultz, Roland; Heberling, Gwendolyn; Görzen, Eugen; Meyer, Hanna; Seeber, Elke; Pinkert, Stefan; Ritz, Markus; Ansorge, Hermann; Bendix, Jörg; Seifert, Bernhard; Miehe, Georg; Long, Ruijun; Yang, Yongping; Wesche, Karsten

    2015-04-01

    Aim Animal husbandry is one of the most widespread land use types, and grazing is a key topic in grassland management. A wide range of indicators are employed in grazing assessments and they often yield widely differing estimates on the associated level of degradation threat. Covering Tibet as a large grassland region with long history of pastoralism, we selected representative indicators to test: (1) how grazing responses change along large-scale climatic gradients, and (2) whether their responses to both grazing intensities and local abiotic conditions are congruent. Location Tibetan Plateau Methods Biotic indicators including species and growth form compositions of vascular plants, richness and abundance of small mammals and ants, together with soil nutrients and field spectra were compared in pairs of high and low grazing intensity at 18 sites across large climatic gradients. Altitude, temperature, and precipitation were considered as potentially influential abiotic factors. Responses of indicators to grazing intensity and environmental gradients were explored by multivariate and univariate analyses. Results All indicators responded strongly to environmental changes, but the response patterns and the most influential abiotic factors varied among indicators. Grazing responses showed low overall congruence. Only vegetation cover, soil nutrient concentrations, and spectral indices were sensitive to grazing across large spatial scales. Grazing effects were significant only when local abiotic factors were taken into account. Main conclusions The results imply that grazing assessments require both appropriate indicators and local calibration. Overall, the threat of grassland degradation across the Tibetan Plateau is not as severe as is commonly assumed.

  12. Nonparametric statistical methods for comparing two sites based on data with multiple non-detect limits

    NASA Astrophysics Data System (ADS)

    Millard, Steven P.; Deverel, Steven J.

    1988-12-01

    As concern over the effects of trace amounts of pollutants has increased, so has the need for statistical methods that deal appropriately with data that include values reported as "less than" the detection limit. It has become increasingly common for water quality data to include censored values that reflect more than one detection limit for a single analyte. For such multiply censored data sets, standard statistical methods (for example, to compare analyte concentration in two areas) are not valid. In such cases, methods from the biostatistical field of survival analysis are applicable. Several common two-sample censored data rank tests are explained, and their behaviors are studied via a Monte Carlo simulation in which sample sizes and censoring mechanisms are varied under an assumed lognormal distribution. These tests are applied to shallow groundwater chemistry data from two sites in the San Joaquin Valley, California. The best overall test, in terms of maintained α level, is the normal scores test based on a permutation variance. In cases where the α level is maintained, however, the Peto-Prentice statistic based on an asymptotic variance performs as well or better.

  13. Using multiple ecological assessment techniques to screen for a wetlands reference site

    SciTech Connect

    Smith, B.M.; McNaughton, E.E.; Wagner, A.; Vig, A.; Zinky, L.; McCormick, S.

    1995-12-31

    Previous investigation of the landfill on the Concord Naval Weapons Station, located in a diked wetland, showed that the sediments exhibited elevated levels of inorganic analytes, as compared to national benchmarks, such as the NOAA ER-Ls. The need to identify a reference marsh was recognized because of the likelihood that these levels of inorganics were representative of ambient conditions, rather than associated with releases to the environment. A marsh location was selected that was upgradient of the landfill, and distant from sources of known contamination, such as roadways and railroad tracks. Sediment samples were analyzed for bulk sediment chemistry, toxicity, and benthic community structure. Bulk sediment chemistry results showed elevated concentrations of lead and petroleum-related organic compounds, perhaps associated with a historic pipeline break. Pore water was prepared from sediment collocated with the bulk chemistry samples and tested with the sea urchin development bioassay. Compared to the control, three of four sediment pore water samples were highly inhibitory of normal development. However, the benthic community survey from the same sampling stations showed a health assemblage of organisms, although one station that exhibited visible petroleum contamination also showed reduced abundance. The conflicting results demonstrate the necessity for taking more than one measure in developing the ecological assessment of a site.

  14. Amplified Rate Acceleration by Simultaneous Up-Regulation of Multiple Active Sites in an Endo-Functionalized Porous Capsule.

    PubMed

    Kopilevich, Sivil; Müller, Achim; Weinstock, Ira A

    2015-10-14

    Using the hydrolysis of epoxides in water as a model reaction, the effect of multiple active sites on Michaelis-Menten compliant rate accelerations in a porous capsule is demonstrated. The capsule is a water-soluble Ih-symmetry Keplerate-type complex of the form, [{Mo(VI)6O21(H2O)6}12{Mo(V)2O4(L)}30](42-), in which 12 pentagonal "ligands," {(Mo(VI))Mo(VI)5O21(H2O)6}(6-), are coordinated to 30 dimolybdenum sites, {Mo(V)2O4L}(1+) (L = an endohedrally coordinated η(2)-bound carboxylate anion), resulting in 20 Mo9O9 pores. When "up-regulated" by removal of ca. one-third of the blocking ligands, L, an equal number of dimolybdenum sites are activated, and the newly freed-up space allows for encapsulation of nearly twice as many substrate guests, leading to a larger effective molarity (amplification), and an increase in the rate acceleration (k(cat)/k(uncat)) from 16,000 to an enzyme-like value of 182,800.

  15. Initial success of native grasses is contingent on multiple interactions among exotic grass competition, temporal priority, rainfall and site effects.

    PubMed

    Young, Truman P; Zefferman, Emily P; Vaughn, Kurt J; Fick, Stephen

    2014-12-05

    Ecological communities are increasingly being recognized as the products of contemporary drivers and historical legacies that are both biotic and abiotic. In an attempt to unravel multiple layers of ecological contingency, we manipulated (i) competition with exotic annual grasses, (ii) the timing of this competition (temporal priority in arrival/seeding times) and (iii) watering (simulated rainfall) in a restoration-style planting of native perennial grasses. In addition, we replicated this experiment simultaneously at three sites in north-central California. Native perennial grasses had 73-99 % less cover when planted with exotic annuals than when planted alone, but this reduction was greatly ameliorated by planting the natives 2 weeks prior to the exotics. In a drought year, irrigation significantly reduced benefits of early planting so that these benefits resembled those observed in a non-drought year. There were significant differences across the three sites (site effects and interactions) in (i) overall native cover, (ii) the response of natives to competition, (iii) the strength of the temporal priority effect and (iv) the degree to which supplemental watering reduced priority effects. These results reveal the strong multi-layered contingency that underlies even relatively simple communities.

  16. Initial success of native grasses is contingent on multiple interactions among exotic grass competition, temporal priority, rainfall and site effects

    PubMed Central

    Young, Truman P.; Zefferman, Emily P.; Vaughn, Kurt J.; Fick, Stephen

    2015-01-01

    Ecological communities are increasingly being recognized as the products of contemporary drivers and historical legacies that are both biotic and abiotic. In an attempt to unravel multiple layers of ecological contingency, we manipulated (i) competition with exotic annual grasses, (ii) the timing of this competition (temporal priority in arrival/seeding times) and (iii) watering (simulated rainfall) in a restoration-style planting of native perennial grasses. In addition, we replicated this experiment simultaneously at three sites in north-central California. Native perennial grasses had 73–99 % less cover when planted with exotic annuals than when planted alone, but this reduction was greatly ameliorated by planting the natives 2 weeks prior to the exotics. In a drought year, irrigation significantly reduced benefits of early planting so that these benefits resembled those observed in a non-drought year. There were significant differences across the three sites (site effects and interactions) in (i) overall native cover, (ii) the response of natives to competition, (iii) the strength of the temporal priority effect and (iv) the degree to which supplemental watering reduced priority effects. These results reveal the strong multi-layered contingency that underlies even relatively simple communities. PMID:25480888

  17. Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

    PubMed

    Mets, Toomas; Lippus, Markus; Schryer, David; Liiv, Aivar; Kasari, Villu; Paier, Anton; Maiväli, Ülo; Remme, Jaanus; Tenson, Tanel; Kaldalu, Niilo

    2017-01-02

    The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5'- and 3'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

  18. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

    PubMed

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-08-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats☆

    PubMed Central

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-01-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. PMID:23648487

  20. A Cationic MOF with High Uptake and Selectivity for CO2 due to Multiple CO2 -Philic Sites.

    PubMed

    Wang, Hai-Hua; Shi, Wen-Juan; Hou, Lei; Li, Gao-Peng; Zhu, Zhonghua; Wang, Yao-Yu

    2015-11-09

    The reaction of N-rich pyrazinyl triazolyl carboxyl ligand 3-(4-carboxylbenzene)-5-(2-pyrazinyl)-1H-1,2,4-triazole (H2 cbptz) with MnCl2 afforded 3D cationic metal-organic framework (MOF) [Mn2 (Hcbptz)2 (Cl)(H2 O)]Cl⋅DMF⋅0.5 CH3 CN (1), which has an unusual (3,4)-connected 3,4T1 topology and 1D channels composed of cavities. MOF 1 has a very polar framework that contains exposed metal sites, uncoordinated N atoms, narrow channels, and Cl(-) basic sites, which lead to not only high CO2 uptake, but also remarkably selective adsorption of CO2 over N2 and CH4 at 298-333 K. The multiple CO2 -philic sites were identified by grand canonical Monte Carlo simulations. Moreover, 1 shows excellent stability in natural air environment. These advantages make 1 a very promising candidate in post-combustion CO2 capture, natural-gas upgrading, and landfill gas-purification processes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Fluorouracil cream 0.5% for actinic keratoses on multiple body sites: an 18-month open-label study.

    PubMed

    Stough, Dow; Bucko, Alicia D; Vamvakias, George; Rafal, Elyse S; Davis, Steven A

    2010-05-01

    This prospective 18-month, open-label, multicenter study assessed the long-term safety and efficacy of fluorouracil cream 0.5% in 277 participants with multiple actinic keratoses (AKs) on the face/anterior scalp and other body sites. Two treatment/observation cycles were separated by 12 months. During treatment cycle 1 (TC1), all participants were treated with fluorouracil cream 0.5% for 4 weeks with 4-week follow-up. Twelve months later, all participants were assessed for treatment cycle 2 (TC2); participants with face/anterior scalp AKs (N = 98) were re-treated with fluorouracil cream 0.5% for 4 weeks with 4-week follow-up. Only 4 participants (7.4%) experienced a treatment-related adverse event (AE) that was not an application site reaction or eye irritation. No unexpected AEs were reported; most were mild or moderate. After TC1 (week 8), the number of AK lesions was significantly reduced on the face/anterior scalp and all other treated body sites (P < .0001). Clearance rates were 30.5% (hands), 39.8% (face/anterior scalp), and 79.1% (lips). After TC2 (week 60), face/anterior scalp AKs were significantly reduced (P < .0001) and the clearance rate was 33.3%. This study indicates that fluorouracil cream 0.5% with a patented microsponge delivery system was well-tolerated and effective in treating and preventing recurrence of AK lesions up to 18 months after initial treatment.

  2. Natural selection affects multiple aspects of genetic variation at putatively neutral sites across the human genome.

    PubMed

    Lohmueller, Kirk E; Albrechtsen, Anders; Li, Yingrui; Kim, Su Yeon; Korneliussen, Thorfinn; Vinckenbosch, Nicolas; Tian, Geng; Huerta-Sanchez, Emilia; Feder, Alison F; Grarup, Niels; Jørgensen, Torben; Jiang, Tao; Witte, Daniel R; Sandbæk, Annelli; Hellmann, Ines; Lauritzen, Torsten; Hansen, Torben; Pedersen, Oluf; Wang, Jun; Nielsen, Rasmus

    2011-10-01

    A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations.

  3. Natural Selection Affects Multiple Aspects of Genetic Variation at Putatively Neutral Sites across the Human Genome

    PubMed Central

    Lohmueller, Kirk E.; Albrechtsen, Anders; Li, Yingrui; Kim, Su Yeon; Korneliussen, Thorfinn; Vinckenbosch, Nicolas; Tian, Geng; Huerta-Sanchez, Emilia; Feder, Alison F.; Grarup, Niels; Jørgensen, Torben; Jiang, Tao; Witte, Daniel R.; Sandbæk, Annelli; Hellmann, Ines; Lauritzen, Torsten; Hansen, Torben

    2011-01-01

    A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations. PMID:22022285

  4. Sequence analysis of Plasmodium falciparum cytochrome b in multiple geographic sites

    PubMed Central

    Ekala, Marie-Thérèse; Khim, Nimol; Legrand, Eric; Randrianarivelojosia, Milijaona; Jambou, Ronan; Fandeur, Thierry; Menard, Didier; Assi, Serge-Brice; Henry, Marie-Claire; Rogier, Christophe; Bouchier, Christiane; Mercereau-Puijalon, Odile

    2007-01-01

    Background The antimalarial drug atovaquone specifically targets Plasmodium falciparum cytochrome b (Pfcytb), a mitochondrial gene with uniparental inheritance. Cases of resistance to atovaquone associated with mutant Pfcytb have been reported, justifying efforts to better document the natural polymorphism of this gene. To this end, a large molecular survey was conducted in several malaria endemic areas where atovaquone was not yet in regular use. Methods The polymorphism of the Pfcytb was analysed by direct sequencing of PCR products corresponding to the full length coding region. Sequence was generated for 671 isolates originating from three continents: Africa (Senegal, Ivory Coast, Central African Republic and Madagascar), Asia (Cambodia) and South America (French Guiana). Results Overall, 11 polymorphic sites were observed, of which eight were novel mutations. There was a large disparity in the geographic distribution of the mutants. All isolates from Senegal, Central African Republic and Madagascar displayed a Camp/3D7 wild type Pfcytb sequence, as did most samples originating from Cambodia and Ivory Coast. One synonymous (t759a at codon V253V) and two non-synonymous (t553g and a581g at codons F185V and H194R, respectively) singletons were detected in Ivory Coast. Likewise, two synonymous (a126t and c793t at codons -T42T and L265L, respectively) singletons were observed in Cambodia. In contrast, seven mutated sites, affecting seven codons and defining four mutant haplotypes were observed in French Guiana. The wild type allele was observed in only 14% of the French Guiana isolates. The synonymous c688t mutation at position L230L was highly prevalent; the most frequent allele was the c688t single mutant, observed in 84% of the isolates. The other alleles were singletons (a126t/a165c, a4g/a20t/a1024c and a20t/t341c/c688t corresponding to T42T/S55S, N2D/N71I/I342L, N71I/L114S/L230L, respectively" please replace with ' corresponding to T42T/S55S, N2D/N71I/I342L and

  5. Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria.

    PubMed

    Zhang, Can; Wang, Yuanchao; Sun, Huzhi; Ren, Huiying

    2015-10-01

    The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

  6. Pharmacophore modeling using site-identification by ligand competitive saturation (SILCS) with multiple probe molecules.

    PubMed

    Yu, Wenbo; Lakkaraju, Sirish Kaushik; Raman, E Prabhu; Fang, Lei; MacKerell, Alexander D

    2015-02-23

    Receptor-based pharmacophore modeling is an efficient computer-aided drug design technique that uses the structure of the target protein to identify novel leads. However, most methods consider protein flexibility and desolvation effects in a very approximate way, which may limit their use in practice. The Site-Identification by Ligand Competitive Saturation (SILCS) assisted pharmacophore modeling protocol (SILCS-Pharm) was introduced recently to address these issues, as SILCS naturally takes both protein flexibility and desolvation effects into account by using full molecular dynamics simulations to determine 3D maps of the functional group-affinity patterns on a target receptor. In the present work, the SILCS-Pharm protocol is extended to use a wider range of probe molecules including benzene, propane, methanol, formamide, acetaldehyde, methylammonium, acetate and water. This approach removes the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor probe molecule. The new SILCS-Pharm protocol is shown to yield improved screening results, as compared to the previous approach based on three target proteins. Further validation of the new protocol using five additional protein targets showed improved screening compared to those using common docking methods, further indicating improvements brought by the explicit inclusion of additional feature types associated with the wider collection of probe molecules in the SILCS simulations. The advantage of using complementary features and volume constraints, based on exclusion maps of the protein defined from the SILCS simulations, is presented. In addition, reranking using SILCS-based ligand grid free energies is shown to enhance the diversity of identified ligands for the majority of targets. These results suggest that the SILCS-Pharm protocol will be of utility in rational drug design.

  7. Interaction between CME and surrounding magnetic fields producing multiple flaring sites

    NASA Astrophysics Data System (ADS)

    van Driel-Gesztelyi, Lidia M.

    2015-08-01

    L. van Driel-Gesztelyi (1,2,3), D. Baker (1), T. Török (4), E. Pariat (2), L.M. Green (1),D.R. Williams (1), J. Carlyle (1,5) G. Valori (1, 2), P. Démoulin (2), B. Kliem (1,7,8),D. Long (1), S.A. Matthews (1), J.-M. Malherbe (2)(1) UCL/MSSL, UK, (2) Paris Observatory, LESIA, CNRS, France, (3) Konkoly Observatory, Hungary, (4) Predictive Science, Dan Diego, USA, (5) Max Planck Inst., Göttingen, Germany, (6) INAF, Obs. Roma, Italy, (7) Potsdam Univ., Germany, (8) Yunnan Observatories, Kunming, ChinaAnalyzing Solar Dynamics Observatory (SDO) observations of the spectacular Coronal Mass Ejection eruption on 7 June 2011, we present evidence of coronal magnetic reconnection between the expanding magnetic structure of the CME and the magnetic fields of an adjacent active region (AR). The onset of reconnection first became apparent in the SDO/AIA images when filament plasma, originally contained within the erupting flux rope, was re-directed towards remote areas in the neighboring AR, tracing the change of large-scale magnetic connectivity. The observations are presented jointly with a topological analysis of the pre-eruption magnetic configuration, and a data-constrained numerical simulation of the three-AR complex, demonstrating the formation/intensification of current sheets along a pre-existing hyperbolic flux tube (HFT) at the interface between the CME and the neighboring AR, where a secondary flare ribbon was created. Reconnection across this current sheet resulted in the formation of new magnetic connections between the erupting magnetic structure and a neighboring AR about 200 Mm from the eruption site, in strong qualitative agreement with the observations. In addition, the CME temporarily created unusually dense plasma conditions around a reconnection region at high coronal altitudes, enabling us to observe emission resulting from it. We argue that this exceptional observation of a coronal brightening was directly observable at SDO/AIA wavelengths owing to the

  8. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

    PubMed Central

    Grandori, C; Mac, J; Siëbelt, F; Ayer, D E; Eisenman, R N

    1996-01-01

    The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism. Images PMID:8861962

  9. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-01-01

    In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a

  10. Minimization of self-quenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores

    PubMed Central

    Zhegalova, Natalia G.; He, Shawn; Zhou, Haiying; Kim, David M.; Berezin, Mikhail Y.

    2014-01-01

    Self-aggregation of dyes even at low concentrations pose a considerable challenge in preparing sufficiently bright molecular probes for in vivo imaging, particularly in the conjugation of near infrared cyanine dyes to polypeptides with multiple labeling sites. Such self-aggregation leads to a significant energy transfer between the dyes resulting in severe quenching and low brightness of the targeted probe. To address this problem, we designed a novel type of dye with an asymmetrical distribution of charge. Asymmetrical distribution prevents the chromophores from π-stacking thus minimizing the energy transfer and fluorescence quenching. The conjugation of the dye to polypeptides showed only a small presence of an H-aggregate band in the absorption spectra and, hence, a relatively high quantum efficiency. PMID:24764130

  11. An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells.

    PubMed

    Koh, Esther Y C; Ho, Steven C L; Mariati; Song, Zhiwei; Bi, Xuezhi; Bardor, Muriel; Yang, Yuansheng

    2013-01-01

    A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10(th), 11(th), and 12(th) AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

  12. Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast.

    PubMed

    Takahashi, Tatsuro; Ohara, Eri; Nishitani, Hideo; Masukata, Hisao

    2003-02-17

    In most eukaryotes, replication origins are composed of long chromosome regions, and the exact sequences required for origin recognition complex (ORC) and minichromosome maintenance (MCM) complex association remain elusive. Here, we show that two stretches of adenine/thymine residues are collectively essential for a fission yeast chromosomal origin. Chromatin immunoprecipitation assays revealed that the ORC subunits are located within a 1 kb region of ori2004. Analyses of deletion derivatives of ori2004 showed that adenine stretches are required for ORC binding in vivo. Synergistic interaction between ORC and adenine stretches was observed. On the other hand, MCM subunits were localized preferentially to a region near the initiation site, which is distant from adenine stretches. This association was dependent on adenine stretches and stimulated by a non-adenine element. Our results suggest that association of multiple ORC molecules with a replication origin is required for efficient MCM loading and origin firing in fission yeast.

  13. Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo

    PubMed Central

    2011-01-01

    Background Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions. Results Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems. Conclusions Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes. PMID

  14. Mini-Tn7 Insertion in Bacteria With Multiple glmS-Linked attTn7 Sites: Example Burkholderia Mallei ATCC 23344

    DTIC Science & Technology

    2006-06-27

    bacterial gen- omes contain single glmS genes, and therefore most probably only a single attTn7 site. With the possible exception of Burkholderia glumae ...mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkholderia mallei ATCC 23344 Kyoung-Hee Choi1, David DeShazer2...downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we

  15. Oxygen Vacancy-Tuned Physical Properties in Perovskite Thin Films with Multiple B-site Valance States

    NASA Astrophysics Data System (ADS)

    Enriquez, Erik; Chen, Aiping; Harrell, Zach; Dowden, Paul; Koskelo, Nicholas; Roback, Joseph; Janoschek, Marc; Chen, Chonglin; Jia, Quanxi

    2017-04-01

    Controlling oxygen content in perovskite oxides with ABO3 structure is one of most critical steps for tuning their functionality. Notably, there have been tremendous efforts to understand the effect of changes in oxygen content on the properties of perovskite thin films that are not composed of cations with multiple valance states. Here, we study the effect of oxygen vacancies on structural and electrical properties in epitaxial thin films of SrFeO3-δ (SFO), where SFO is a compound with multiple valance states at the B site. Various annealing treatments are used to produce different oxygen contents in the films, which has resulted in significant structural changes in the fully strained SFO films. The out-of-plane lattice parameter and tetragonality increase with decreasing oxygen concentration, indicating the crystal structure is closely related to the oxygen content. Importantly, variation of the oxygen content in the films significantly affects the dielectric properties, leakage conduction mechanisms, and the resistive hysteresis of the materials. These results establish the relationship between oxygen content and structural and functional properties for a range of multivalent transition metal oxides.

  16. Oxygen Vacancy-Tuned Physical Properties in Perovskite Thin Films with Multiple B-site Valance States

    PubMed Central

    Enriquez, Erik; Chen, Aiping; Harrell, Zach; Dowden, Paul; Koskelo, Nicholas; Roback, Joseph; Janoschek, Marc; Chen, Chonglin; Jia, Quanxi

    2017-01-01

    Controlling oxygen content in perovskite oxides with ABO3 structure is one of most critical steps for tuning their functionality. Notably, there have been tremendous efforts to understand the effect of changes in oxygen content on the properties of perovskite thin films that are not composed of cations with multiple valance states. Here, we study the effect of oxygen vacancies on structural and electrical properties in epitaxial thin films of SrFeO3−δ (SFO), where SFO is a compound with multiple valance states at the B site. Various annealing treatments are used to produce different oxygen contents in the films, which has resulted in significant structural changes in the fully strained SFO films. The out-of-plane lattice parameter and tetragonality increase with decreasing oxygen concentration, indicating the crystal structure is closely related to the oxygen content. Importantly, variation of the oxygen content in the films significantly affects the dielectric properties, leakage conduction mechanisms, and the resistive hysteresis of the materials. These results establish the relationship between oxygen content and structural and functional properties for a range of multivalent transition metal oxides. PMID:28417954

  17. Oxygen Vacancy-Tuned Physical Properties in Perovskite Thin Films with Multiple B-site Valance States

    DOE PAGES

    Enriquez, Erik; Chen, Aiping; Harrell, Zach; ...

    2017-04-18

    Controlling oxygen content in perovskite oxides with ABO3 structure is one of most critical steps for tuning their functionality. Notably, there have been tremendous efforts to understand the effect of changes in oxygen content on the properties of perovskite thin films that are not composed of cations with multiple valance states. Here, we study the effect of oxygen vacancies on structural and electrical properties in epitaxial thin films of SrFeO3-δ (SFO), where SFO is a compound with multiple valance states at the B site. Various annealing treatments are used to produce different oxygen contents in the films, which has resultedmore » in significant structural changes in the fully strained SFO films. The out-of-plane lattice parameter and tetragonality increase with decreasing oxygen concentration, indicating the crystal structure is closely related to the oxygen content. Importantly, variation of the oxygen content in the films significantly affects the dielectric properties, leakage conduction mechanisms, and the resistive hysteresis of the materials. These results establish the relationship between oxygen content and structural and functional properties for a range of multivalent transition metal oxides.« less

  18. Primary care approaches to musculoskeletal multiple-site joint pain pharmacological therapy: a survey of general practitioners.

    PubMed

    Raja, Rafi; Kingsbury, Sarah R; Wise, Elspeth; Conaghan, Philip G

    2014-10-01

    Chronic multiple-site joint pain (MSJP) due to osteoarthritis and soft tissue disorders is common in people over 50 years old and associated with poor outcomes. This study examined current pharmacological approaches to MSJP management in primary care. One hundred and fifty general practitioners (GPs) attending an educational seminar participated in an electronic survey (mean response rate 96%). Most GPs reported treating multiple painful joints concurrently (78%) compared with focusing on a single joint (21%). The majority believed there was no difference in analgesia for different disorders when selecting paracetamol (84%), non-steroidal anti-inflammatory drugs (NSAID)/COX-2 inhibitors (57%) or opioids (70%). When optimising therapy, intra-class optimisation (increase NSAID dose 41%, change to another NSAID/COX-2 inhibitor 30%) was preferred to inter-class step up therapy (add opioid 23%, change to opioid 6%). For NSAID gastrointestinal intolerance, the preference was to add a gastro-protective agent (74%). There is a need to better characterise MSJP and examine optimal pharmacotherapy regimens.

  19. S4MPLE--Sampler for Multiple Protein-Ligand Entities: Methodology and Rigid-Site Docking Benchmarking.

    PubMed

    Hoffer, Laurent; Chira, Camelia; Marcou, Gilles; Varnek, Alexandre; Horvath, Dragos

    2015-05-19

    This paper describes the development of the unified conformational sampling and docking tool called Sampler for Multiple Protein-Ligand Entities (S4MPLE). The main novelty in S4MPLE is the unified dealing with intra- and intermolecular degrees of freedom (DoF). While classically programs are either designed for folding or docking, S4MPLE transcends this artificial specialization. It supports folding, docking of a flexible ligand into a flexible site and simultaneous docking of several ligands. The trick behind it is the formal assimilation of inter-molecular to intra-molecular DoF associated to putative inter-molecular contact axes. This is implemented within the genetic operators powering a Lamarckian Genetic Algorithm (GA). Further novelty includes differentiable interaction fingerprints to control population diversity, and fitting a simple continuum solvent model and favorable contact bonus terms to the AMBER/GAFF force field. Novel applications-docking of fragment-like compounds, simultaneous docking of multiple ligands, including free crystallographic waters-were published elsewhere. This paper discusses: (a) methodology, (b) set-up of the force field energy functions and (c) their validation in classical redocking tests. More than 80% success in redocking was achieved (RMSD of top-ranked pose < 2.0 Å).

  20. Bivalent Compound 17MN Exerts Neuroprotection through Interaction at Multiple Sites in a Cellular Model of Alzheimer's Disease.

    PubMed

    Liu, Kai; Chojnacki, Jeremy E; Wade, Emily E; Saathoff, John M; Lesnefsky, Edward J; Chen, Qun; Zhang, Shijun

    2015-01-01

    Multiple pathogenic factors have been suggested to play a role in the development of Alzheimer's disease (AD). The multifactorial nature of AD also suggests the potential use of compounds with polypharmacology as effective disease-modifying agents. Recently, we have developed a bivalent strategy to include cell membrane anchorage into the molecular design. Our results demonstrated that the bivalent compounds exhibited multifunctional properties and potent neuroprotection in a cellular AD model. Herein, we report the mechanistic exploration of one of the representative bivalent compounds, 17MN, in MC65 cells. Our results established that MC65 cells die through a necroptotic mechanism upon the removal of tetracycline (TC). Furthermore, we have shown that mitochondrial membrane potential and cytosolic Ca2+ levels are increased upon removal of TC. Our bivalent compound 17MN can reverse such changes and protect MC65 cells from TC removal induced cytotoxicity. The results also suggest that 17MN may function between the Aβ species and RIPK1 in producing its neuroprotection. Colocalization studies employing a fluorescent analog of 17MN and confocal microscopy demonstrated the interactions of 17MN with both mitochondria and endoplasmic reticulum, thus suggesting that 17MN exerts its neuroprotection via a multiple-site mechanism in MC65 cells. Collectively, these results strongly support our original design rationale of bivalent compounds and encourage further optimization of this bivalent strategy to develop more potent analogs as novel disease-modifying agents for AD.

  1. A non-independent energy-based multiple sequence alignment improves prediction of transcription factor binding sites.

    PubMed

    Salama, Rafik A; Stekel, Dov J

    2013-11-01

    Multiple sequence alignments (MSAs) are usually scored under the assumption that the sequences being aligned have evolved by common descent. Consequently, the differences between sequences reflect the impact of insertions, deletions and mutations. However, non-coding DNA binding sequences, such as transcription factor binding sites (TFBSs), are frequently not related by common descent, and so the existing alignment scoring methods are not well suited for aligning such sequences. We present a novel multiple MSA methodology that scores TFBS DNA sequences by including the interdependence of neighboring bases. We introduced two variants supported by different underlying null hypotheses, one statistically and the other thermodynamically generated. We assessed the alignments through their performance in TFBS prediction; both methods show considerable improvements when compared with standard MSA algorithms. Moreover, the thermodynamically generated null hypothesis outperforms the statistical one due to improved stability in the base stacking free energy of the alignment. The thermodynamically generated null hypothesis method can be downloaded from http://sourceforge.net/projects/msa-edna/. dov.stekel@nottingham.ac.uk. Supplementary data are available at Bioinformatics online.

  2. Temperature dependent CO2 behavior in microporous 1-D channels of a metal-organic framework with multiple interaction sites

    PubMed Central

    Kim, Dongwook; Park, Jaehun; Kim, Yung Sam; Lah, Myoung Soo

    2017-01-01

    The MOF with the encapsulated CO2 molecule shows that the CO2 molecule is ligated to the unsaturated Cu(II) sites in the cage using its Lewis basic oxygen atom via an angular η1-(OA) coordination mode and also interacts with Lewis basic nitrogen atoms of the tetrazole ligands using its Lewis acidic carbon atom. Temperature dependent structure analyses indicate the simultaneous weakening of both interactions as temperature increases. Infrared spectroscopy of the MOF confirmed that the CO2 interaction with the framework is temperature dependent. The strength of the interaction is correlated to the separation of the two bending peaks of the bound CO2 rather than the frequency shift of the asymmetric stretching peak from that of free CO2. The encapsulated CO2 in the cage is weakly interacting with the framework at around ambient temperatures and can have proper orientation for wiggling out of the cage through the narrow portals so that the reversible uptake can take place. On the other hand, the CO2 in the cage is restrained at a specific orientation at 195 K since it interacts with the framework strong enough using the multiple interaction sites so that adsorption process is slightly restricted and desorption process is almost clogged. PMID:28128298

  3. Temperature dependent CO2 behavior in microporous 1-D channels of a metal-organic framework with multiple interaction sites

    NASA Astrophysics Data System (ADS)

    Kim, Dongwook; Park, Jaehun; Kim, Yung Sam; Lah, Myoung Soo

    2017-01-01

    The MOF with the encapsulated CO2 molecule shows that the CO2 molecule is ligated to the unsaturated Cu(II) sites in the cage using its Lewis basic oxygen atom via an angular η1-(OA) coordination mode and also interacts with Lewis basic nitrogen atoms of the tetrazole ligands using its Lewis acidic carbon atom. Temperature dependent structure analyses indicate the simultaneous weakening of both interactions as temperature increases. Infrared spectroscopy of the MOF confirmed that the CO2 interaction with the framework is temperature dependent. The strength of the interaction is correlated to the separation of the two bending peaks of the bound CO2 rather than the frequency shift of the asymmetric stretching peak from that of free CO2. The encapsulated CO2 in the cage is weakly interacting with the framework at around ambient temperatures and can have proper orientation for wiggling out of the cage through the narrow portals so that the reversible uptake can take place. On the other hand, the CO2 in the cage is restrained at a specific orientation at 195 K since it interacts with the framework strong enough using the multiple interaction sites so that adsorption process is slightly restricted and desorption process is almost clogged.

  4. Source apportionment of PM2.5 at multiple sites in Venice (Italy): Spatial variability and the role of weather

    NASA Astrophysics Data System (ADS)

    Masiol, Mauro; Squizzato, Stefania; Rampazzo, Giancarlo; Pavoni, Bruno

    2014-12-01

    This study investigates the chemical speciation of fine particulate matter (PM2.5) collected at three sites in the Venice area, eastern Po Valley (Italy). This area is one of the few hot spots left in Europe where levels of PM2.5 frequently breach EU target values and cause a serious risk for public health. Elemental composition, inorganic ions and polycyclic aromatic hydrocarbon concentrations were quantified in 448 PM2.5 samples and the multiple-site PMF receptor model was based on the elemental and inorganic ion data. Six factors associated with potential sources were quantified, namely, secondary sulfate, ammonium nitrate and combustions, fossil fuels, traffic, industrial and glassmaking. Source apportionment results were further processed using a series of chemometric tools for returning additional information about the seasonal and spatial changes of factors extracted by the PMF analysis. In addition, PMF results were also studied in combination with weather conditions and PAH concentrations revealing that sources of secondary nitrate and sulfate are homogeneously distributed throughout the area, while remaining pollutant sources may have a distinct origin. PMF results were cluster analyzed to sort out samples with similar source profiles and then the wind roses of grouped samples were examined to assess the role of wind speed and direction on PM2.5 pollution and chemistry. The tested tools and the results obtained can be used for air quality assessment studies and air pollution reduction strategies.

  5. Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites*

    PubMed Central

    Bellanca, Sebastiano; Summers, Robert L.; Meyrath, Max; Dave, Anurag; Nash, Megan N.; Dittmer, Martin; Sanchez, Cecilia P.; Stein, Wilfred D.; Martin, Rowena E.; Lanzer, Michael

    2014-01-01

    Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance. PMID:25378409

  6. Serotype-Specific Changes in Invasive Pneumococcal Disease after Pneumococcal Conjugate Vaccine Introduction: A Pooled Analysis of Multiple Surveillance Sites

    PubMed Central

    Feikin, Daniel R.; Kagucia, Eunice W.; Loo, Jennifer D.; Link-Gelles, Ruth; Puhan, Milo A.; Cherian, Thomas; Levine, Orin S.; Whitney, Cynthia G.; O’Brien, Katherine L.; Moore, Matthew R.

    2013-01-01

    Background Vaccine-serotype (VT) invasive pneumococcal disease (IPD) rates declined substantially following introduction of 7-valent pneumococcal conjugate vaccine (PCV7) into national immunization programs. Increases in non-vaccine-serotype (NVT) IPD rates occurred in some sites, presumably representing serotype replacement. We used a standardized approach to describe serotype-specific IPD changes among multiple sites after PCV7 introduction. Methods and Findings Of 32 IPD surveillance datasets received, we identified 21 eligible databases with rate data ≥2 years before and ≥1 year after PCV7 introduction. Expected annual rates of IPD absent PCV7 introduction were estimated by extrapolation using either Poisson regression modeling of pre-PCV7 rates or averaging pre-PCV7 rates. To estimate whether changes in rates had occurred following PCV7 introduction, we calculated site specific rate ratios by dividing observed by expected IPD rates for each post-PCV7 year. We calculated summary rate ratios (RRs) using random effects meta-analysis. For children <5 years old, overall IPD decreased by year 1 post-PCV7 (RR 0·55, 95% CI 0·46–0·65) and remained relatively stable through year 7 (RR 0·49, 95% CI 0·35–0·68). Point estimates for VT IPD decreased annually through year 7 (RR 0·03, 95% CI 0·01–0·10), while NVT IPD increased (year 7 RR 2·81, 95% CI 2·12–3·71). Among adults, decreases in overall IPD also occurred but were smaller and more variable by site than among children. At year 7 after introduction, significant reductions were observed (18–49 year-olds [RR 0·52, 95% CI 0·29–0·91], 50–64 year-olds [RR 0·84, 95% CI 0·77–0·93], and ≥65 year-olds [RR 0·74, 95% CI 0·58–0·95]). Conclusions Consistent and significant decreases in both overall and VT IPD in children occurred quickly and were sustained for 7 years after PCV7 introduction, supporting use of PCVs. Increases in NVT IPD occurred in most sites, with variable magnitude

  7. Serotype-specific changes in invasive pneumococcal disease after pneumococcal conjugate vaccine introduction: a pooled analysis of multiple surveillance sites.

    PubMed

    Feikin, Daniel R; Kagucia, Eunice W; Loo, Jennifer D; Link-Gelles, Ruth; Puhan, Milo A; Cherian, Thomas; Levine, Orin S; Whitney, Cynthia G; O'Brien, Katherine L; Moore, Matthew R

    2013-01-01

    Vaccine-serotype (VT) invasive pneumococcal disease (IPD) rates declined substantially following introduction of 7-valent pneumococcal conjugate vaccine (PCV7) into national immunization programs. Increases in non-vaccine-serotype (NVT) IPD rates occurred in some sites, presumably representing serotype replacement. We used a standardized approach to describe serotype-specific IPD changes among multiple sites after PCV7 introduction. Of 32 IPD surveillance datasets received, we identified 21 eligible databases with rate data ≥ 2 years before and ≥ 1 year after PCV7 introduction. Expected annual rates of IPD absent PCV7 introduction were estimated by extrapolation using either Poisson regression modeling of pre-PCV7 rates or averaging pre-PCV7 rates. To estimate whether changes in rates had occurred following PCV7 introduction, we calculated site specific rate ratios by dividing observed by expected IPD rates for each post-PCV7 year. We calculated summary rate ratios (RRs) using random effects meta-analysis. For children <5 years old, overall IPD decreased by year 1 post-PCV7 (RR 0.55, 95% CI 0.46-0.65) and remained relatively stable through year 7 (RR 0.49, 95% CI 0.35-0.68). Point estimates for VT IPD decreased annually through year 7 (RR 0.03, 95% CI 0.01-0.10), while NVT IPD increased (year 7 RR 2.81, 95% CI 2.12-3.71). Among adults, decreases in overall IPD also occurred but were smaller and more variable by site than among children. At year 7 after introduction, significant reductions were observed (18-49 year-olds [RR 0.52, 95% CI 0.29-0.91], 50-64 year-olds [RR 0.84, 95% CI 0.77-0.93], and ≥ 65 year-olds [RR 0.74, 95% CI 0.58-0.95]). Consistent and significant decreases in both overall and VT IPD in children occurred quickly and were sustained for 7 years after PCV7 introduction, supporting use of PCVs. Increases in NVT IPD occurred in most sites, with variable magnitude. These findings may not represent the experience in low-income countries or the

  8. Radius bone strength in bending, compression, and falling and its correlation with clinical densitometry at multiple sites.

    PubMed

    Lochmüller, Eva-Maria; Lill, Christoph A; Kuhn, Volker; Schneider, Erich; Eckstein, Felix

    2002-09-01

    This study comprehensively analyzes the ability of site-specific and nonsite-specific clinical densitometric techniques for predicting mechanical strength of the distal radius in different loading configurations. DXA of the distal forearm, spine, femur, and total body and peripheral quantitative computed tomography (pQCT) measurements of the distal radius (4, 20, and 33%) were obtained in situ (with soft tissues) in 129 cadavers, aged 80.16 +/- 9.8 years. Spinal QCT and calcaneal quantitative ultrasound (QUS) were performed ex situ in degassed specimens. The left radius was tested in three-point bending and axial compression, and the right forearm was tested in a fall configuration, respectively. Correlation coefficients with radius DXA were r = 0.89, 0.84, and 0.70 for failure in three-point bending, axial compression, and the fall simulation, respectively. The correlation with pQCT (r = 0.75 for multiple regression models with the fall) was not significantly higher than for DXA. Nonsite-specific measurements and calcaneal QUS displayed significantly (p < 0.01) lower correlation coefficients, and QUS did only contribute to the prediction of axial failure stress but not of failure load. We conclude that a combination of pQCT parameters involves only marginal improvement in predicting mechanical strength of the distal radius, nonsite-specific measurements are less accurate for this purpose, and QUS adds only little independent information to site-specific bone mass. Therefore, the noninvasive diagnosis of loss of strength at the distal radius should rely on site-specific measurements with DXA or pQCT and may be the earliest chance to detect individuals at risk of osteoporotic fracture.

  9. The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites*

    PubMed Central

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-01-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats). PMID:25792750

  10. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    PubMed

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The value of testing multiple anatomic sites for gonorrhoea and chlamydia in sexually transmitted infection centres in the Netherlands, 2006-2010.

    PubMed

    Koedijk, F D H; van Bergen, J E A M; Dukers-Muijrers, N H T M; van Leeuwen, A P; Hoebe, C J P A; van der Sande, M A B

    2012-09-01

    National surveillance data from 2006 to 2010 of the Dutch sexually transmitted infection (STI) centres were used to analyse current practices on testing extragenital sites for chlamydia and gonorrhoea in men who have sex with men (MSM) and women. In MSM, 76.0% and 88.9% were tested at least at one extragenital site (pharyngeal and/or anorectal) for chlamydia and gonorrhoea, respectively; for women this was 20.5% and 30.2%. Testing more than one anatomic site differed by STI centre, ranging from 2% to 100%. In MSM tested at multiple sites, 63.0% and 66.5% of chlamydia and gonorrhoea diagnoses, respectively, would have been missed if screened at the urogenital site only, mainly anorectal infections. For women tested at multiple sites, the proportions of missed chlamydia and gonorrhoea diagnoses would have been 12.9% and 30.0%, respectively. Testing extragenital sites appears warranted, due to the numerous infections that would have been missed. Adding anorectal screening to urogenital screening for all MSM visiting an STI centre should be recommended. Since actual testing practices differ by centre, there is a need for clearer guidelines. Routine gonorrhoea and chlamydia screening at multiple sites in STI centres should be investigated further as this might be a more effective approach to reduce transmission than current practice.

  12. Fast IPSPs elicited via multiple synaptic release sites by different types of GABAergic neurone in the cat visual cortex.

    PubMed Central

    Tamás, G; Buhl, E H; Somogyi, P

    1997-01-01

    cells established multiple synaptic junctions on their postsynaptic target cells. A basket cell innervated a pyramidal cell via fifteen release sites; the numbers of synapses formed by three dendrite-targeting cells on pyramidal cells were seventeen and eight respectively, and three on a spiny stellate cell; the interaction between a double bouquet cell and a postsynaptic pyramidal cell was mediated by ten synaptic junctions. 5. All three types of interneurone (n = 6; 2 for each type of cell) elicited short-latency IPSPs with fast rise time (10-90%; 2.59 +/- 1.02 ms) and short duration (at half-amplitude, 15.82 +/- 5.24 ms), similar to those mediated by GABAA receptors. 6. Average amplitudes of unitary IPSPs (n = 6) were 845 +/- 796 microV (range, 134-2265 microV). Variability of IPSP amplitude was moderate, the average ratio of IPSP and baseline noise variance was 1.54 +/- 0.96. High frequency activation of single presynaptic dendrite-targeting cells led to an initial summation followed by use-dependent depression of the averaged postsynaptic response. Double bouquet cell-evoked IPSPs, recorded in the soma, had a smaller amplitude than those evoked by the other two cell types. In all connections, transmission failures were rare or absent, particularly when mediated by a high number of release sites. 7. The results demonstrate that different types of neocortical GABAergic neurones innervate distinct domains on the surface of their postsynaptic target cells. Nevertheless, all three types of cell studied here elicit fast IPSPs and provide GABAergic input through multiple synaptic release sites with few, if any, failures of transmission. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9161987

  13. Progression of a Fracture Site Impaction as a Prognostic Indicator of Impacted Femoral Neck Fracture Treated with Multiple Pinning

    PubMed Central

    Yoon, Pil Whan; Shin, Young Ho; Yoo, Jeong Joon; Yoon, Kang Sup

    2012-01-01

    Background We evaluated the clinical and radiologic results of impacted femoral neck fractures treated with multiple pinning and determined the influence of the progression of impaction at the fracture site on clinical outcome. Methods There were 34 patients with a mean age of 65.5 years. The mean follow-up period was 3.4 years. Progression of fracture site impaction was measured using an articulo-trochanteric distance index and the percentage decrease in the articulo-trochanteric distance index between follow-up intervals. The failure of treatment was clarified as non-union and avascular necrosis. Other characteristics of the patients, including mean waiting time for surgery, preoperative Singh index score, and body mass index, were also measured to evaluate the influence on the clinical outcome of surgery. Results There were 6 fractures which were not treated successfully (3 non-union, 8.8% and 3 avascular necrosis, 8.8%). The mean percentage decrease of the articulo-trochanteric distance index within the first 6 weeks after surgery was 4.5% in the successful group and 25.1% in the failure group (p < 0.001). There was also a significant mean percentage decrease in the articulo-trochanteric distance index between 6 weeks and 3 months (p < 0.001). Conclusions Primary stabilization with Knowles pins for impacted femoral neck fractures had a reasonable clinical outcome with low morbidity. Despite a significant difference of a mean percentage decrease in the articulo-trochanteric distance index between the successful group and the failure group, we could not verify it as a risk factor for failure of treatment because the odds ratio was not statistically significant. PMID:22379557

  14. Target site pharmacokinetics of linezolid after single and multiple doses in diabetic patients with soft tissue infection.

    PubMed

    Eslam, Roza Badr; Burian, Angela; Vila, Greisa; Sauermann, Robert; Hammer, Alexandra; Frenzel, Dorothea; Minichmayr, Iris K; Kloft, Charlotte; Matzneller, Peter; Oesterreicher, Zoe; Zeitlinger, Markus

    2014-09-01

    The underlying pathology of diabetic wounds, i.e. impairment of macro- and microcirculation, might also impact target site penetration of antibacterial drugs. To compare tissue concentrations of linezolid in infected and not infected tissue 10 patients suffering from type 2 diabetes with foot infection were included in the study. Tissue penetration of linezolid was assessed using in vivo microdialysis at the site of infection as well as in non-inflamed subcutaneous adipose tissue. All patients were investigated after receiving a single dose of linezolid and five patients in addition at steady state. After a single dose of linezolid significantly higher area under the concentration vs. time curve over 8 hours (AUC0-8 ) and maximum concentrations (Cmax )-values were observed in plasma (65.5 ± 21.2 mg*h/L and 16.4 ± 4.6 mg/L) as compared to inflamed (36.3 ± 22.9  mg*h/L and 6.6 ± 3.6 mg/L) and non-inflamed tissue (33.0 ± 17.7 mg*h/L and 6.7 ± 3.6 mg/L). Multiple administrations of linezolid led to disappearance of significant differences in Cmax and AUC0-8 between plasma, inflamed, and non-inflamed tissue. Approximately 2-fold increase of Cmax and AUC0-8 -values in tissue was observed at steady state as compared to the first administration. Penetration of linezolid is not impaired in diabetic foot infection but equilibrium between plasma and tissue might be delayed.

  15. Dimethylnitrosamine-demethylase: absence of increased enzyme catabolism and multiplicity of effector sites in repression. Hemoprotein involvement.

    PubMed

    Argus, M F; Arcos, J C; Pastor, K M; Wu, B C; Venkatesan, N

    1976-05-01

    Evidence is presented that the previously observed decrease of the Vmax of hepatic microsomal demethylation of dimethylnitrosamine (DMN), following pretreatment of rats with 3-methylcholanthrene (MC), is not due to increase in the rate of breakdown but to decrease of de novo synthesis. Determinations of Vmax at time intervals in the transition from the high steady-state level induced by a carbohydrate-devoid casein diet, down to the low steady-state level of carbohydrate-containing basal diet, yielded two consecutive slopes; descent from the basal diet level to the lower steady-state level following pretreatment with MC yielded one slope. Plotting these slopes against the initial Vmax values gave a typical exponential curve (or straight line if the logs of slopes are used) indicating that the rate of enzyme decay in the MC-treated animals is not greater than that expected from normal enzyme catabolism. A multiplicity of effector sites appears to be involved in the repressor action of different structural types; for example, repression by MC (46.6%) and by phenobarbital (23.9%) in combination are approximately additive (62.0%), rather than competitive, indicating that the two agents act at different sites. A P-450 type cytochrome is involved in the demethylation of DMN. DMN-demethylase is inhibited by carbon monoxide, but the susceptibility to CO is far greater than that observed previously with 3,4-benzopyrene hydroxylation; inhibition of DMN-demethylase as a function of CO concentration follows typical enzyme kinetics. However, while both phenobarbital and MC powerfully repress the DMN-demethylase, we have confirmed that they are strong inducers of the synthesis of P-450 and P-448, respectively, as estimated from the difference spectra.

  16. Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites.

    PubMed

    Wadsworth, Caroline; Procopio, Noemi; Anderung, Cecilia; Carretero, José-Miguel; Iriarte, Eneko; Valdiosera, Cristina; Elburg, Rengert; Penkman, Kirsty; Buckley, Michael

    2017-03-31

    Ancient DNA (aDNA) is the most informative biomolecule extracted from skeletal remains at archaeological sites, but its survival is unpredictable and its extraction and analysis is time consuming, expensive and often fails. Several proposed methods for better understanding aDNA survival are based upon the characterisation of some aspect of protein survival, but these are typically non-specific; proteomic analyses may offer an attractive method for understanding preservation processes. In this study, in-depth proteomic (LC-Orbitrap-MS/MS) analyses were carried out on 69 archaeological bovine bone and dentine samples from multiple European archaeological sites and compared with mitochondrial aDNA and amino acid racemisation (AAR) data. Comparisons of these data, including estimations of the relative abundances for seven selected non-collagenous proteins, indicate that the survival of aDNA in bone or dentine may correlate with the survival of some proteins, and that proteome complexity is a more useful predictor of aDNA survival than protein abundance or AAR. The lack of a strong correlation between the recovery of aDNA and the proteome abundance may indicate that the survival of aDNA is more closely linked to its ability to associate with bone hydroxyapatite crystals rather than to associate with proteins. Ancient biomolecule survival remains poorly understood, even with great advancements in 'omics' technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be. Copyright © 2017. Published by Elsevier B.V.

  17. Progression of a fracture site impaction as a prognostic indicator of impacted femoral neck fracture treated with multiple pinning.

    PubMed

    Yoon, Pil Whan; Shin, Young Ho; Yoo, Jeong Joon; Yoon, Kang Sup; Kim, Hee Joong

    2012-03-01

    We evaluated the clinical and radiologic results of impacted femoral neck fractures treated with multiple pinning and determined the influence of the progression of impaction at the fracture site on clinical outcome. There were 34 patients with a mean age of 65.5 years. The mean follow-up period was 3.4 years. Progression of fracture site impaction was measured using an articulo-trochanteric distance index and the percentage decrease in the articulo-trochanteric distance index between follow-up intervals. The failure of treatment was clarified as non-union and avascular necrosis. Other characteristics of the patients, including mean waiting time for surgery, preoperative Singh index score, and body mass index, were also measured to evaluate the influence on the clinical outcome of surgery. There were 6 fractures which were not treated successfully (3 non-union, 8.8% and 3 avascular necrosis, 8.8%). The mean percentage decrease of the articulo-trochanteric distance index within the first 6 weeks after surgery was 4.5% in the successful group and 25.1% in the failure group (p < 0.001). There was also a significant mean percentage decrease in the articulo-trochanteric distance index between 6 weeks and 3 months (p < 0.001). Primary stabilization with Knowles pins for impacted femoral neck fractures had a reasonable clinical outcome with low morbidity. Despite a significant difference of a mean percentage decrease in the articulo-trochanteric distance index between the successful group and the failure group, we could not verify it as a risk factor for failure of treatment because the odds ratio was not statistically significant.

  18. The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

    PubMed Central

    Hubber, Andree; Arasaki, Kohei; Nakatsu, Fubito; Hardiman, Camille; Lambright, David; De Camilli, Pietro; Nagai, Hiroki; Roy, Craig R.

    2014-01-01

    The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis. PMID:24992562

  19. Multiple techniques for mineral identification on Mars:. a study of hydrothermal rocks as potential analogues for astrobiology sites on Mars

    NASA Astrophysics Data System (ADS)

    Bishop, Janice L.; Murad, Enver; Lane, Melissa D.; Mancinelli, Rocco L.

    2004-06-01

    Spectroscopic studies of Mars analog materials combining multiple spectral ranges and techniques are necessary in order to obtain ground truth information for interpretation of rocks and soils on Mars. Two hydrothermal rocks from Yellowstone National Park, Wyoming, were characterized here because they contain minerals requiring water for formation and they provide a possible niche for some of the earliest organisms on Earth. If related rocks formed in hydrothermal sites on Mars, identification of these would be important for understanding the geology of the planet and potential habitability for life. XRD, thermal properties, VNIR, mid-IR, and Raman spectroscopy were employed to identify the mineralogy of the samples in this study. The rocks studied here include a travertine from Mammoth Formation that contains primarily calcite with some aragonite and gypsum and a siliceous sinter from Octopus Spring that contains a variety of poorly crystalline to amorphous silicate minerals. Calcite was detected readily in the travertine rock using any one of the techniques studied. The small amount of gypsum was uniquely identified using XRD, VNIR, and mid-IR, while the aragonite was uniquely identified using XRD and Raman. The siliceous sinter sample was more difficult to characterize using each of these techniques and a combination of all techniques was more useful than any single technique. Although XRD is the historical standard for mineral identification, it presents some challenges for remote investigations. Thermal properties are most useful for minerals with discrete thermal transitions. Raman spectroscopy is most effective for detecting polarized species such as CO 3, OH, and CH, and exhibits sharp bands for most highly crystalline minerals when abundant. Mid-IR spectroscopy is most useful in characterizing Si-O (and metal-O) bonds and also has the advantage that remote information about sample texture (e.g., particle size) can be determined. Mid-IR spectroscopy is also

  20. Compact Solar Spectrometer Column CO2, and CH4 Observations: Performance Evaluation at Multiple North American TCCON Sites

    NASA Astrophysics Data System (ADS)

    Parker, H. A.; Hedelius, J.; Viatte, C.; Wunch, D.; Wennberg, P. O.; Chen, J.; Wofsy, S.; Jones, T.; Franklin, J.; Dubey, M. K.; Roehl, C. M.; Podolske, J. R.; Hillyard, P. W.; Iraci, L. T.

    2015-12-01

    Measurement, reporting and verification (MRV) of anthropogenic emissions and natural sources and sinks of carbon dioxide (CO2) and methane (CH4) are crucial to predict climate change and develop transparent accounting policies to contain climate forcing. Remote sensing technologies are monitoring column averaged dry air mole fractions of CO2 and CH4 (XCO2 & XCH4) from ground and space (OCO-2 and GOSAT) with solar spectroscopy enabling direct MRV. However, current ground based coverage is sparse due to the need for large and expensive high-resolution spectrometers that are part of the Total Column Carbon Observing Network (TCCON, Bruker 125HR). This limits our MRV and satellite validation abilities, both regionally and globally. There are striking monitoring gaps in Asia, South America and Africa where the CO2 emissions are growing and there is a large uncertainty in fluxes from land use change, biomass burning and rainforest vulnerability. To fill this gap we evaluate the precision, accuracy and stability of compact, affordable and easy to use low-resolution spectrometers (Bruker EM27/SUN) by comparing with XCO2 and XCH4 retrieved from much larger high-resolution TCCON instruments. As these instruments will be used in a variety of locations, we evaluate their performance by comparing with 2 previous and 4 current United States TCCON sites in different regions up to 2700 km apart. These sites range from polluted to unpolluted, latitudes of 32 to 46°N, and altitudes of 230 to 2241 masl. Comparisons with some of these sites cover multiple years allowing assessment of the EM27/SUN performance not only in various regions, but also over an extended period of time and with different seasonal influences. Results show that our 2 EM27/SUN instruments capture the diurnal variability of the aforementioned constituents very well, but with offsets from TCCON and long-term variability which may be due in part to the extensive movement these spectrometers were subjected to. These

  1. Phenyl aldehyde and propanoids exert multiple sites of action towards cell membrane and cell wall targeting ergosterol in Candida albicans

    PubMed Central

    2013-01-01

    In the present study, two phyto-compounds phenyl aldehyde (cinnamaldehyde) and propanoid (eugenol) were selected to explore their modes of action against Candida albicans. Electron microscopy, flow cytometry and spectroscopic assays were employed to determine the targets of these compounds. Treatment of C. albicans (CA04) with sub-MICs of cinnamaldehyde (50 μg/mL) and eugenol (200 μg/mL) indicated multiple sites of action including damages to cell walls, cell membranes, cytoplasmic contents and other membranous structures as observed under electron microscopy. Concentration and time dependent increase in the release of cytoplasmic contents accompanied with change in extracellular K+ concentration was recorded. Exposure of Candida cells at 4 × MIC of cinnaamldehyde and eugenol resulted in 40.21% and 50.90% dead cells, respectively as revealed by flow cytometry analysis. Treatment of Candida cells by cinnamaldehyde and eugenol at 0.5 × MIC showed 67.41% and 76.23% reduction in ergosterol biosynthesis, respectively. The binding assays reflected the ability of compounds to bind with the ergosterol. Our findings have suggested that the membrane damaging effects of phenyl aldehyde and propanoids class of compounds is attributed to their ability to inhibit ergosterol biosynthesis and simultaneously binding with ergosterol. Indirect or secondary action of these compounds on cell wall is also expected as revealed by electron microscopic studies. PMID:24010721

  2. Mitochondrial motility in axons: membranous organelles may interact with the force generating system through multiple surface binding sites.

    PubMed

    Martz, D; Lasek, R J; Brady, S T; Allen, R D

    1984-01-01

    In living tissue, membrane-bound organelles, including mitochondria, move along parallel cytoplasmic pathways. Motion is directed and tends to be confined to a single path. Deviations from this single path motion are rare. When present, however, they tend to occur at points of intersection of cytoskeletal linear elements (LE). Such intersections are relatively uncommon in intact axons and extruded axoplasm. However, we have found that such intersections can be produced in extruded preparations by shear forces directed tangential to the axoplasmic surface. We have studied the detailed behavior of mitochondria in extruded squid axoplasm. Special attention was directed to the relationship between mitochondrial shape changes and orientation of cytoskeletal LE. The most striking of these changes in shape is branching. In this process, the mitochondrion transiently assumes a triradial (three-ended) shape. This appearance may be maintained for seconds to minutes before the normal cylindrical shape is resumed by absorption of either the newly formed end or, more commonly, one of the original ends. The frequency of branching appears to be dependent on the degree of cytoskeletal organization. It becomes more common as the number of apparent intersections between cytoskeletal LE increases. Further, the formation of new ends seems to occur along paths defined by cytoskeletal elements. These observations suggest that the mitochondrial membrane is multivalent. That is, it contains multiple sites capable of interacting with the axonal force generation apparatus. Furthermore, LE in the cytoskeleton may indicate the paths along which these interactions are permissible.

  3. Intracranial meningeal hemangiopericytoma: Recurrences at the initial and distant intracranial sites and extraneural metastases to multiple organs.

    PubMed

    Wei, Guangquan; Kang, Xiaowei; Liu, Xianping; Tang, Xing; Li, Qinlong; Han, Juntao; Yin, Hong

    2015-07-01

    Regardless of the controversial pathogenesis, intracranial meningeal hemangiopericytoma (M-HPC) is a rare, highly cellular and vascularized mesenchymal tumor that is characterized by a high tendency for recurrence and extraneural metastasis, despite radical excision and postoperative radiotherapy. M-HPC shares similar clinical manifestations and radiological findings with meningioma, which causes difficulty in differentiation of this entity from those prognostically favorable mimics prior to surgery. Treatment of M-HPC, particularly in metastatic settings, remains a challenge. A case is described of primary M-HPC with recurrence at the initial and distant intracranial sites and extraneural multiple-organ metastases in a 36-year-old female. The metastasis of M-HPC was extremely extensive, and to the best of our knowledge this is the first case of M-HPC with delayed metastasis to the bilateral kidneys. The data suggests that preoperative computed tomography and magnetic resonance imaging could provide certain diagnostic clues and useful information for more optimal treatment planning. The results may imply that novel drugs, such as temozolomide and bevacizumab, as a component of multimodality therapy of M-HPC may deserve further investigation.

  4. Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export.

    PubMed

    Port, Sarah A; Monecke, Thomas; Dickmanns, Achim; Spillner, Christiane; Hofele, Romina; Urlaub, Henning; Ficner, Ralf; Kehlenbach, Ralph H

    2015-10-27

    CRM1 is the major nuclear export receptor. During translocation through the nuclear pore, transport complexes transiently interact with phenylalanine-glycine (FG) repeats of multiple nucleoporins. On the cytoplasmic side of the nuclear pore, CRM1 tightly interacts with the nucleoporin Nup214. Here, we present the crystal structure of a 117-amino-acid FG-repeat-containing fragment of Nup214, in complex with CRM1, Snurportin 1, and RanGTP at 2.85 Å resolution. The structure reveals eight binding sites for Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor. Nup214 binds to N- and C-terminal regions of CRM1, thereby clamping CRM1 in a closed conformation and stabilizing the export complex. The role of conserved hydrophobic pockets for the recognition of FG motifs was analyzed in biochemical and cell-based assays. Comparative studies with RanBP3 and Nup62 shed light on specificities of CRM1-nucleoporin binding, which serves as a paradigm for transport receptor-nucleoporin interactions.

  5. Assembly of ROMK1 (Kir 1.1a) inward rectifier K+ channel subunits involves multiple interaction sites.

    PubMed

    Koster, J C; Bentle, K A; Nichols, C G; Ho, K

    1998-04-01

    The ROMK1 (Kir 1.1a) channel is formed by a tetrameric complex of subunits, each characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment. To delineate the general regions mediating the assembly of ROMK1 subunits we constructed epitope-tagged N-terminal, C-terminal, and transmembrane segment deletion mutants. Nonfunctional subunits with N-terminal, core region, and C-terminal deletions had dominant negative effects when coexpressed with wild-type ROMK1 subunits in Xenopus oocytes. In contrast, coexpression of these nonfunctional subunits with Kv 2.1 (DRK1) did not suppress Kv 2.1 currents in control oocytes. Interactions between epitope-tagged mutant and wild-type ROMK1 subunits were studied in parallel by immunoprecipitating [35S]-labeled oocyte membrane proteins. Complexes containing both wild-type and mutant subunits that retained H5, M2, and C-terminal regions were coimmunoprecipitated to a greater extent than complexes consisting of wild-type and mutant subunits with core region and/or C-terminal deletions. The present findings are consistent with the hypothesis that multiple interaction sites located in the core region and cytoplasmic termini of ROMK1 subunits mediate homomultimeric assembly.

  6. Assembly of ROMK1 (Kir 1.1a) inward rectifier K+ channel subunits involves multiple interaction sites.

    PubMed Central

    Koster, J C; Bentle, K A; Nichols, C G; Ho, K

    1998-01-01

    The ROMK1 (Kir 1.1a) channel is formed by a tetrameric complex of subunits, each characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment. To delineate the general regions mediating the assembly of ROMK1 subunits we constructed epitope-tagged N-terminal, C-terminal, and transmembrane segment deletion mutants. Nonfunctional subunits with N-terminal, core region, and C-terminal deletions had dominant negative effects when coexpressed with wild-type ROMK1 subunits in Xenopus oocytes. In contrast, coexpression of these nonfunctional subunits with Kv 2.1 (DRK1) did not suppress Kv 2.1 currents in control oocytes. Interactions between epitope-tagged mutant and wild-type ROMK1 subunits were studied in parallel by immunoprecipitating [35S]-labeled oocyte membrane proteins. Complexes containing both wild-type and mutant subunits that retained H5, M2, and C-terminal regions were coimmunoprecipitated to a greater extent than complexes consisting of wild-type and mutant subunits with core region and/or C-terminal deletions. The present findings are consistent with the hypothesis that multiple interaction sites located in the core region and cytoplasmic termini of ROMK1 subunits mediate homomultimeric assembly. PMID:9545044

  7. Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

    SciTech Connect

    Baskaran, Sulochanadevi; Chikwana, Vimbai M.; Contreras, Christopher J.; Davis, Keri D.; Wilson, Wayne A.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D.

    2012-12-10

    Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V{sub max}/S{sub 0.5} for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V{sub max}/S{sub 0.5} for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells ({Delta}gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a 'toehold mechanism,' keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.

  8. Systematic discovery of molecular probes targeting multiple non-orthosteric and spatially distinct sites in the botulinum neurotoxin subtype A (BoNT/A).

    PubMed

    Dadgar, Saedeh; Floriano, Wely B

    2015-06-01

    The development of molecular probes targeting proteins has traditionally relied on labeling compounds already known to bind to the protein of interest. These known ligands bind to orthosteric or allosteric sites in their target protein as a way to control their activity. Binding pockets other than known orthosteric or allosteric sites may exist that are large enough to accommodate a ligand without significantly disrupting protein activity. Such sites may provide opportunities to discriminate between subtypes or other closely related proteins, since they are under less evolutionary pressure to be conserved. The Protein Scanning with Virtual Ligand Screening (PSVLS) approach was previously used to identify a novel inhibitor and a fluorescent probe against the catalytic site of the botulinum neurotoxin subtype A (BoNT/A). PSVLS screens compound databases against multiple sites within a target protein, and the results for all the sites probed against BoNT/A, not only the catalytic site, are available online. Here, we analyze the PSVLS data for multiple sites in order to identify molecular probes with affinity for binding pockets other than the catalytic site of BoNT/A. BoNT/A is a large protein with a light (LC) and a heavy (HC) chain that can be assayed separately. We used scintillation proximity assay (SPA) to test experimentally 5 probe candidates predicted computationally to have affinity for different non-orthosteric binding regions within the HC and LC, and one compound predicted not to have affinity for either domain. The binding profiles obtained experimentally confirmed the targeting of multiple and spatially distinct pockets within BoNT/A. Moreover, inhibition assay results indicate that some of these probes do not significantly interfere with the catalytic activity of BoNT/A.

  9. Integrating ecosystems measurements from multiple eddy-covariance sites to a simple model of ecosystem process - Are there possibilities for a uniform model calibration?

    NASA Astrophysics Data System (ADS)

    Minunno, Francesco; Peltoniemi, Mikko; Launiainen, Samuli; Mäkelä, Annikki

    2014-05-01

    Biogeochemical models quantify the material and energy flux exchanges between biosphere, atmosphere and soil, however there is still considerable uncertainty underpinning model structure and parametrization. The increasing availability of data from of multiple sources provides useful information for model calibration and validation at different space and time scales. We calibrated a simplified ecosystem process model PRELES to data from multiple sites. In this work we had the following objective: to compare a multi-site calibration and site-specific calibrations, in order to test if PRELES is a model of general applicability, and to test how well one parameterization can predict ecosystem fluxes. Model calibration and evaluation were carried out by the means of the Bayesian method; Bayesian calibration (BC) and Bayesian model comparison (BMC) were used to quantify the uncertainty in model parameters and model structure. Evapotranspiration (ET) and gross primary production (GPP) measurements collected in 9 sites of Finland and Sweden were used in the study; half dataset was used for model calibrations and half for the comparative analyses. 10 BCs were performed; the model was independently calibrated for each of the nine sites (site-specific calibrations) and a multi-site calibration was achieved using the data from all the sites in one BC. Then 9 BMCs were carried out, one for each site, using output from the multi-site and the site-specific versions of PRELES. Similar estimates were obtained for the parameters at which model outputs are most sensitive. Not surprisingly, the joint posterior distribution achieved through the multi-site calibration was characterized by lower uncertainty, because more data were involved in the calibration process. No significant differences were encountered in the prediction of the multi-site and site-specific versions of PRELES, and after BMC, we concluded that the model can be reliably used at regional scale to simulate carbon and

  10. X-ray radiation and the risk of multiple sclerosis: Do the site and dose of exposure matter?

    PubMed Central

    Motamed, Mohammad Reza; Fereshtehnejad, Seyed-Mohammad; Abbasi, Maryam; Sanei, Mastaneh; Abbaslou, Mina; Meysami, Somayeh

    2014-01-01

    Background: The sporadic cases of radiation-activated multiple sclerosis (MS) has been previously described, with a few studies focused on the relationship between radiation and the risk of MS. The aim of our study was to evaluate the association between history of X-ray radiation and MS. Methods: This case-control study was conducted on 150 individuals including 65 MS patients and 85 age- and sex-matched healthy controls enrolled using non-probability convenient sampling. Any history of previous Xray radiation consisted of job-related X-ray exposure, radiotherapy, radiographic evaluations including chest Xray, lumbosacral X-ray, skull X-ray, paranasal sinuses (PNS) X-ray, gastrointestinal (GI) series, foot X-ray and brain CT scanning were recorded and compared between two groups. Statistical analysis was performed using independent t test, Chi square and receiver operating characteristics (ROC) curve methods through SPSS software. Results: History of both diagnostic [OR=3.06 (95% CI: 1.32-7.06)] and therapeutic [OR=7.54 (95% CI: 1.5935.76) X-ray radiations were significantly higher among MS group. Mean number of skull X-rays [0.4 (SD=0.6) vs. 0.1 (SD=0.3), p=0.004] and brain CT scanning [0.9 (SD=0.8) vs. 0.5 (SD=0.7), p=0.005] was higher in MS group as well as mean of the cumulative X-ray radiation dosage [1.84 (SD=1.70) mSv vs. 1.11 (SD=1.54) mSv; p=0.008]. Conclusion: Our study was one of the first to show higher history of X-ray radiation in patients with MS compared to healthy controls. A possible association was also found between the dose and the site exposed to X-ray radiation and risk of developing MS PMID:25695003

  11. Multiple Natural Hazards Assessment and Comparison to Planned Land Use in an Andean Touristic Site within the Riskscape Central Chile

    NASA Astrophysics Data System (ADS)

    Braun, Andreas; Jaque Castillo, Edilia

    2017-04-01

    The Andes of central Chile are a natural environment characterized by multiple natural hazards (mass movements, volcanic hazards, seismic hazards, snow avalanches to name a few). The totality of these hazards, according to the notion of Müller-Mahn et al. an in relation to vulnerable entities, spans a riskscape. Spatial planning should take this riskscape into account in order to ensure a save an resilient regional development. However, as frequently observed in developing or newly developed countries, such precaution measures are only hardly realized. Spatial planing tends to be reactive to private inversion, opportunistic and frequently clientelistic. This results in spatial structures whose future development is vulnerable to natural disasters. The contribution analyses these circumstances within a riskscape in central Chile. Within the VIII. Region, close to the volcanic complex Nevados de Chillan, a touristic development around a Hotel for winter sports is established. However, the place is affected by a multitude of natural hazards. The contribution, on the basis of primary and secondary data, first provides hazard maps for several natural hazards. Secondly, the individual hazard maps are merged to an overall hazard map. This overall hazard map is related to the vulnerable entities to span a riskscape. The vulnerable entities are settlements, but also tourist infrastructures. Then, the contribution compares how a precautions spatial planning could have avoided putting vulnerable entities at risk, which spatial structure - especially regarding tourism - is actually found and which challenges for spatial development do exist. It reveals that the most important tourist infrastructures are found particularly at places, characterized by a high overall hazard. Furthermore, it will show that alternatives at economically equally attractive sites, but with a much smaller overall hazard, would have existed. It concludes by discussing possible reasons for this by

  12. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    SciTech Connect

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  13. Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

    SciTech Connect

    D'Amato, R.J.; Largent, B.L.; Snowman, A.M.; Snyder, S.H.

    1987-07-01

    Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine binding reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.

  14. Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

    PubMed Central

    Mitchell, P J; Carothers, A M; Han, J H; Harding, J D; Kas, E; Venolia, L; Chasin, L A

    1986-01-01

    Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream. Images PMID:3023846

  15. Multiple muscle-specific regulatory elements are associated with a DNase I hypersensitive site of the cardiac beta-myosin heavy-chain gene.

    PubMed

    Huang, W Y; Chen, J J; Shih, N; Liew, C C

    1997-10-15

    Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5'-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region. We further localized the beta-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the beta-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the beta-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the beta-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the beta-MyHC gene may be associated with the beta-2.3 kb site.

  16. Comparison of single and multiple dwell position methods in MammoSite high dose rate (HDR) brachytherapy planning.

    PubMed

    Kim, Yongbok; Trombetta, Mark G; Miften, Moyed

    2010-05-28

    The purpose of this study is to dosimetrically compare two plans generated using single dwell position method (SDPM) and multiple dwell position methods (MDPM) in MammoSite high dose rate (HDR) brachytherapy planning for 19 breast cancer patients. In computed tomography (CT) image-based HDR planning, a surface optimization technique was used in both methods. Following dosimetric parameters were compared for fraction 1 plans: %PTV_EVAL (planning target volume for plan evaluation) coverage, dose homogeneity index (DHI), dose con-formal index (COIN), maximum dose to skin and ipsilateral lung, and breast tissue volume receiving 150% (V150[cc]) and 200% (V200[cc]) of the prescribed dose. In addition, a plan was retrospectively generated for each fraction 2-10 to simulate the clinical situation where the fraction 1 plan was used for fractions 2-10 without modification. In order to create nine derived plans for each method and for each of the 19 patients, the catheter location and contours of target and critical structures were defined on the CT images acquired prior to each fraction 2-10, while using the same dwell-time distribution as used for fraction 1 (original plan). Interfraction dose variations were evaluated for 19 patients by comparing the derived nine plans (each for fractions 2-10) with the original plan (fraction 1) using the same dosimetric parameters used for fraction 1 plan comparison. For the fraction 1 plan comparison, the MDPM resulted in slightly increased %PTV_EVAL coverage, COIN, V150[cc] and V200[cc] values by an average of 1.2%, 0.025, 0.5 cc and 0.7cc, respectively, while slightly decreased DHI, maximum skin and ipsilateral lung dose by an average of 0.003, 3.2 cGy and 5.8 cGy, respectively. For the inter-fraction dose variation comparison, the SDPM resulted in slightly smaller variations in %PTV_EVAL coverage, DHI, maximum skin dose and V150[cc] values by an average of 0.4%, 0.0005, 0.5 cGy and 0.2 cc, respectively, while slightly higher average

  17. A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates

    PubMed Central

    2013-01-01

    Background Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated. Results Screening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser309-Lys334) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase. Conclusions Our work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications. PMID:24161150

  18. Geologic, hydrologic, and water-quality data from multiple-well monitoring sites in the Central and West Coast basins, Los Angeles County, California, 1995-2000

    USGS Publications Warehouse

    Land, Michael; Everett, R.R.; Crawford, S.M.

    2002-01-01

    In 1995, the U.S. Geological Survey (USGS), in cooperation with the HYPERLINK 'http://wrd.org' Water Replenishment District of Southern California (WRDSC), began a study to examine ground-water resources in the Central and West Coast Basins in Los Angeles County, California. The study characterizes the geohydrology and geochemistry of the regional ground-water flow system and provides extensive data for evaluating ground-water management issues. This report is a compilation of geologic, hydrologic, and water-quality data collected from 24 recently constructed multiple-well monitoring sites for the period 1995?2000. Descriptions of the collected drill cuttings were compiled into lithologic logs, which are summarized along with geophysical logs?including gamma-ray, spontaneous potential, resistivity, electromagnetic induction, and temperature tool logs?for each monitoring site. At selected sites, cores were analyzed for magnetic orientation, physical and thermal properties, and mineralogy. Field and laboratory estimates of hydraulic conductivity are presented for most multiple-well monitoring sites. Periodic water-level measurements are also reported. Water-quality information for major ions, nutrients, trace elements, deuterium and oxygen-18, and tritium is presented for the multiple-well monitoring locations, and for selected existing production and observation wells. In addition, boron-11, carbon-13, carbon-14, sulfur-34, and strontium-87/86 data are presented for selected wells.

  19. Moving towards multiple site outcomes in spinal cord injury pain clinical trials: An issue of clustered observations in trial design and analysis.

    PubMed

    Richardson, Elizabeth; Redden, David T

    2014-05-01

    Pain remains a problem for many with spinal cord injury (SCI), and there is a need for sound, randomized clinical trials examining the efficacy of existing and novel therapeutics. SCI-related pain is complex, as more than one type of pain is often experienced. The purpose of this report is to (i) demonstrate how to design and power calculation of a clinical trial of SCI pain using multiple pain sites per individual; (ii) discuss consequences of failing to adjust for this; and (iii) provide intraclass correlation (ICC) estimates for common pain outcome measures that may be used to power future clinical trials in SCI pain. Using an existing dataset from a past SCI pain clinical trial, the ICC was calculated for common pain outcome measures to illustrate appropriate corrections for powering, analyzing and interpreting results from multiple pain sites per individual. The problem associated with not accounting for multiple pain sites per individual and the effect on the Type I error rate is also shown. Not accounting for the ICC can lead to (1) incorrect power estimates in the design of a trial, and (2) an inflated Type I error rate with a higher likelihood of misinterpretation of outcomes. Powering for future SCI pain trials and statistical analysis of trial outcomes may be substantially compromised if methods do not account for the intra-individual associations between pain sites, ultimately affecting study interpretations and evidence-based practice. We present ICC estimates based on SCI pain data for purposes of estimating power for future trials.

  20. Source apportionment of PM2.5 at multiple Northwest U.S. sites: Assessing regional winter wood smoke impacts from residential wood combustion

    NASA Astrophysics Data System (ADS)

    Kotchenruther, Robert A.

    2016-10-01

    Wood smoke from residential wood combustion is a significant source of elevated PM2.5 in many communities across the Northwest U.S. Accurate representation of residential wood combustion in source-oriented regional scale air quality models is challenging because of multiple uncertainties. As an alternative to source-oriented source apportionment, this work provides, through receptor-oriented source apportionment, an assessment of winter residential wood combustion impacts at multiple Northwest U.S. locations. Source apportionment was performed on chemically speciated PM2.5 from 19 monitoring sites using the Positive Matrix Factorization (PMF) receptor model. Each site was modeled independently, but a common data preparation and modeling protocol was used so that results were as comparable as possible across sites. Model solutions had from 4 to 8 PMF factors, depending on the site. PMF factors at each site were associated with a source classification (e.g., primary wood smoke), a dominant chemical composition (e.g., ammonium nitrate), or were some mixture. 15 different sources or chemical compositions were identified as contributing to PM2.5 across the 19 sites. The 6 most common were; aged wood smoke and secondary organic carbon, motor vehicles, primary wood smoke, ammonium nitrate, ammonium sulfate, and fugitive dust. Wood smoke was identified at every site, with both aged and primary wood smoke identified at most sites. Wood smoke contributions to PM2.5 were averaged for the two winter months of December and January, the months when wood smoke in the Northwest U.S. is mainly from residential wood combustion. The total contribution of residential wood combustion, that from primary plus aged smoke, ranged from 11.4% to 92.7% of average December and January PM2.5 depending on the site, with the highest percent contributions occurring in smaller towns that have fewer expected sources of winter PM2.5. Receptor modeling at multiple sites, such as that conducted in this

  1. Studies of the biogenic amine transporters. IV. Demonstration of a multiplicity of binding sites in rat caudate membranes for the cocaine analog [125I]RTI-55.

    PubMed

    Rothman, R B; Cadet, J L; Akunne, H C; Silverthorn, M L; Baumann, M H; Carroll, F I; Rice, K C; de Costa, B R; Partilla, J S; Wang, J B

    1994-07-01

    membranes prepared from rat caudate or COS cells that transiently expressed the cloned cocaine-sensitive DA transporter complementary DNA. Similar experiments also resolved two components of the caudate 5-HT transporter. Viewed collectively, these data provide evidence that [125I]RTI-55 labels multiple binding sites associated with the DA and 5-HT transporters.

  2. Scheduling the Remediation of Port Hope: Logistical and Regulatory Challenges of a Multiple Site Urban Remediation Project - 13119

    SciTech Connect

    Ferguson Jones, Andrea; Lee, Angela; Palmeter, Tim

    2013-07-01

    The Port Hope Project is part of the larger CAN$1.28 billion Port Hope Area Initiative (PHAI), a community-based program for the development and implementation of a safe, local, long-term management solution for historic Low-Level Radioactive Waste (LLRW) in the Municipalities of Port Hope and Clarington, Ontario, Canada. Atomic Energy of Canada (AECL) is the Project Proponent, Public Works and Government Services (PWGSC) is managing the procurement of services and the MMM Group Limited - Conestoga Rovers and Associates Joint Venture (MMM-CRA Joint Venture) is providing detailed design and construction oversight and administration services for the Project. The Port Hope Project includes the construction of a long-term waste management facility (LTWMF) in the Municipality of Port Hope and the remediation of 18 (eighteen) large-scale LLRW, numerous small-scale sites still being identified and industrial sites within the Municipality. The total volume to be remediated is over one million cubic metres and will come from sites that include temporary storage sites, ravines, beaches, parks, private commercial and residential properties and vacant industrial sites all within the urban area of Port Hope. Challenges that will need to be overcome during this 10 year project include: - Requirements stipulated by the Environmental Assessment (EA) that affect Project logistics and schedule. - Coordination of site remediation with the construction schedule at the LTWMF. - Physical constraints on transport routes and at sites affecting production rates. - Despite being an urban undertaking, seasonal constrains for birds and fish (i.e., nesting and spawning seasons). - Municipal considerations. - Site-specific constraints. - Site interdependencies exist requiring consideration in the schedule. Several sites require the use of an adjacent site for staging. (authors)

  3. High-risk human papillomavirus infection involving multiple anatomic sites of the female lower genital tract: a multiplex real-time polymerase chain reaction-based study.

    PubMed

    Hui, Yiang; Manna, Pradip; Ou, Joyce J; Kerley, Spencer; Zhang, Cunxian; Sung, C James; Lawrence, W Dwayne; Quddus, M Ruhul

    2015-09-01

    High-risk human papillomavirus infection usually is seen at one anatomic site in an individual. Rarely, infection at multiple anatomic sites of the female lower genital tract in the same individual is encountered either simultaneously and/or at a later date. The current study identifies the various subtypes of high-risk human papillomavirus infection in these scenarios and analyzes the potential significance of these findings. High-risk human papillomavirus infection involving 22 anatomic sites from 7 individuals was identified after institutional review board approval. Residual paraffin-embedded tissue samples were retrieved, and all 15 high-risk human papillomavirus were identified and viral load quantified using multiplex real-time polymerase chain reaction-based method. Multiple high-risk human papillomavirus subtypes were identified in 32% of the samples and as many as 5 different subtypes of high-risk human papillomavirus infection in a single anatomic site. In general, each anatomic site has unique combination of viral subtypes, although one individual showed overlapping subtypes in the vagina, cervix, and vulvar samples. Higher viral load and rare subtypes are more frequent in younger patients and in dysplasia compared with carcinoma. Follow-up ranging from 3 to 84 months revealed persistent high-risk human papillomavirus infection in 60% of cases. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. [[superscript 3]H]-Flunitrazepam-Labeled Benzodiazepine Binding Sites in the Hippocampal Formation in Autism: A Multiple Concentration Autoradiographic Study

    ERIC Educational Resources Information Center

    Guptill, Jeffrey T.; Booker, Anne B.; Gibbs, Terrell T.; Kemper, Thomas L.; Bauman, Margaret L.; Blatt, Gene J.

    2007-01-01

    Increasing evidence indicates that the GABAergic system in cerebellar and limbic structures is affected in autism. We extended our previous study that found reduced [[superscript 3]H] flunitrazepam-labeled benzodiazepine sites in the autistic hippocampus to determine whether this reduction was due to a decrease in binding site number (B [subscript…

  5. [[superscript 3]H]-Flunitrazepam-Labeled Benzodiazepine Binding Sites in the Hippocampal Formation in Autism: A Multiple Concentration Autoradiographic Study

    ERIC Educational Resources Information Center

    Guptill, Jeffrey T.; Booker, Anne B.; Gibbs, Terrell T.; Kemper, Thomas L.; Bauman, Margaret L.; Blatt, Gene J.

    2007-01-01

    Increasing evidence indicates that the GABAergic system in cerebellar and limbic structures is affected in autism. We extended our previous study that found reduced [[superscript 3]H] flunitrazepam-labeled benzodiazepine sites in the autistic hippocampus to determine whether this reduction was due to a decrease in binding site number (B [subscript…

  6. SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments

    PubMed Central

    Jessen, Leon Eyrich; Hoof, Ilka; Lund, Ole; Nielsen, Morten

    2013-01-01

    Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype–phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying ‘hot’ or ‘cold’ regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype–phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/. PMID:23761454

  7. Soil-water movement under natural-site and waste-site conditions: A multiple-year field study in the Mojave Desert, Nevada

    USGS Publications Warehouse

    Andraski, B.J.

    1997-01-01

    Soil-water movement under natural-site and simulated waste-site conditions were compared by monitoring four experimental sites in the Mojave Desert, Nevada, during a 5-year period: one vegetated soil profile, one soil profile where vegetation was removed, and two nonvegetated test trenches. Precipitation ranged from 14 to 162 mm/yr. Temporal changes in water content measured by neutron probe were limited to the upper 0.5-1 m; values ranged from 0.01 to 0.19 m3/m3. Water potential and temperature were measured by thermocouple psychrometers; 77% remained operable for ???4.5 years. For vegetated soil, precipitation that accumulated in the upper 0.75 m of soil was removed by evapotranspiration: water potentials decreased seasonally by 4 to >8 MPa. During 2 years with below-average precipitation, water potentials below the apparent root zone decreased by 2.3 (1.2-m depth) to 0.4 MPa (5-m depth), and the gradients became predominantly upward. Water potentials then rebounded during 2 years with near-and above-average precipitation, and seasonally variant water: potential gradients were reestablished above the 4.2-m depth. Under nonvegetated Waste-site conditions, data indicated the long-term accumulation and shallow, but continued, penetration of precipitation: water potentials showed moisture penetration to depths of 0.75-1.85 m. The method of simulated-waste drum placement (stacked versus random) and the associated differences m subsidence showed no measurable influence on the water balance of the trenches: subsidence totaled ???13 mm during the study. Water potentials below the trenches and below the 2-m depth ???13 the nonvegetated soil remained low (???-5.5 to -7.5 MPa) and indicated the persistence of typically upward driving forces for isothermal water flow. Water fluxes estimated from water potential and temperature data suggested that isothermal liquid, isothermal vapor, and nonisothermal vapor flow need to be considered in the conceptualization of unsaturated

  8. Evidence for a Non-Catalytic Ion-Binding Site in Multiple RNA-Dependent RNA Polymerases

    PubMed Central

    Mönttinen, Heli A. M.; Ravantti, Janne J.; Poranen, Minna M.

    2012-01-01

    A high-affinity divalent cation-binding site located proximal to the catalytic center has been identified in several RNA-dependent RNA polymerases (RdRps), but the characteristics of such a site have not been systematically studied. Here, all available polymerase structures that follow the hand-like structural motif were screened for the presence of a divalent cation close to the catalytic site but distinct from catalytic metal ions. Such non-catalytic ions were found in all RNA virus families for which there were high-resolution RdRp structures available. Bound ions were always located in structurally similar locations at an approximate 6-Å distance from the catalytic site. Furthermore, the second aspartate residue in the highly conserved GDD sequence was found to be involved in the coordination of the bound ion in all viral RdRps studied. These results suggest that a non-catalytic ion-binding site is conserved across positive-sense, single-stranded, and double-stranded RNA viruses. Interestingly, a non-catalytic ion was also observed in a similar position in the reverse transcriptase of the human immunodeficiency virus. Moreover, two members of the DNA-dependent DNA polymerase B family displayed an ion at a comparable distance from the catalytic site, but the position was clearly distinct from the non-catalytic ion-binding sites of RdRps. PMID:22792374

  9. Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

    SciTech Connect

    Valley, Cary T.; Porter, Douglas F.; Qiu, Chen; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2012-06-28

    mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

  10. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    SciTech Connect

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  11. Multiplicity of carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible evolutionary implications.

    PubMed

    Sharma, Alok; Chandran, Divya; Singh, Desh D; Vijayan, M

    2007-09-01

    The beta-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, beta-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the beta-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a beta-prism I fold lectin, griffthsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of beta-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion.It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the beta-prism II fold, is related to the role of plant lectins in defence.

  12. Processive degradation of unstructured protein by Escherichia coli Lon occurs via the slow, sequential delivery of multiple scissile sites followed by rapid and synchronized peptide bond cleavage events.

    PubMed

    Mikita, Natalie; Cheng, Iteen; Fishovitz, Jennifer; Huang, Jonathan; Lee, Irene

    2013-08-20

    Processive protein degradation is a common feature found in ATP-dependent proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda N protein (λN) to initiate the first kinetic analysis of the proteolytic mechanism of this enzyme. To this end, experiments were designed to determine the timing of three selected scissile sites in λN approaching the proteolytic site of ELon and their subsequent cleavages to gain insight into the mechanism by which ATP-dependent proteases attain processivity in protein degradation. The kinetic profile of peptide bond cleavage at different regions of λN was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic λN constructs were then generated as reporter substrates for transient kinetic characterization of the ATP- versus AMPPNP-dependent peptide bond cleavage and the delivery of the scissile sites near the amino- versus carboxyl-terminal of the λN protein to the proteolytic site of ELon. Collectively, our results support a mechanism by which the cleavage of multiple peptide bonds awaits the "almost complete" delivery of all the scissile sites in λN to the proteolytic site in an ATP-dependent manner. Comparing the time courses of delivery to the active site of the selected scissile sites further implicates the existence of a preferred directionality in the final stage of substrate delivery, which begins at the carboxyl-terminal. The subsequent cleavage of the scissile sites in λN, however, appears to lack a specific directionality and occurs at a much faster rate than the substrate delivery step.

  13. Integration of paleoseismic data from multiple sites to develop an objective earthquake chronology: Application to the Weber segment of the Wasatch fault zone, Utah

    USGS Publications Warehouse

    DuRoss, C.B.; Personius, S.F.; Crone, A.J.; Olig, S.S.; Lund, W.R.

    2011-01-01

    We present a method to evaluate and integrate paleoseismic data from multiple sites into a single, objective measure of earthquake timing and recurrence on discrete segments of active faults. We apply this method to the Weber segment (WS) of the Wasatch fault zone using data from four fault-trench studies completed between 1981 and 2009. After systematically reevaluating the stratigraphic and chronologic data from each trench site, we constructed time-stratigraphic OxCal models that yield site probability density functions (PDFs) of the times of individual earthquakes. We next qualitatively correlated the site PDFs into a segment-wide earthquake chronology, which is supported by overlapping site PDFs, large per-event displacements, and prominent segment boundaries. For each segment-wide earthquake, we computed the product of the site PDF probabilities in common time bins, which emphasizes the overlap in the site earthquake times, and gives more weight to the narrowest, best-defined PDFs. The product method yields smaller earthquake-timing uncertainties compared to taking the mean of the site PDFs, but is best suited to earthquakes constrained by broad, overlapping site PDFs. We calculated segment-wide earthquake recurrence intervals and uncertainties using a Monte Carlo model. Five surface-faulting earthquakes occurred on the WS at about 5.9, 4.5, 3.1, 1.1, and 0.6 ka. With the exception of the 1.1-ka event, we used the product method to define the earthquake times. The revised WS chronology yields a mean recurrence interval of 1.3 kyr (0.7-1.9-kyr estimated two-sigma [2??] range based on interevent recurrence). These data help clarify the paleoearthquake history of the WS, including the important question of the timing and rupture extent of the most recent earthquake, and are essential to the improvement of earthquake-probability assessments for the Wasatch Front region.

  14. Integration of paleoseismic data from multiple sites to develop an objective earthquake chronology: Application to the Weber segment of the Wasatch fault zone, Utah

    USGS Publications Warehouse

    DuRoss, Christopher B.; Personius, Stephen F.; Crone, Anthony J.; Olig, Susan S.; Lund, William R.

    2011-01-01

    We present a method to evaluate and integrate paleoseismic data from multiple sites into a single, objective measure of earthquake timing and recurrence on discrete segments of active faults. We apply this method to the Weber segment (WS) of the Wasatch fault zone using data from four fault-trench studies completed between 1981 and 2009. After systematically reevaluating the stratigraphic and chronologic data from each trench site, we constructed time-stratigraphic OxCal models that yield site probability density functions (PDFs) of the times of individual earthquakes. We next qualitatively correlated the site PDFs into a segment-wide earthquake chronology, which is supported by overlapping site PDFs, large per-event displacements, and prominent segment boundaries. For each segment-wide earthquake, we computed the product of the site PDF probabilities in common time bins, which emphasizes the overlap in the site earthquake times, and gives more weight to the narrowest, best-defined PDFs. The product method yields smaller earthquake-timing uncertainties compared to taking the mean of the site PDFs, but is best suited to earthquakes constrained by broad, overlapping site PDFs. We calculated segment-wide earthquake recurrence intervals and uncertainties using a Monte Carlo model. Five surface-faulting earthquakes occurred on the WS at about 5.9, 4.5, 3.1, 1.1, and 0.6 ka. With the exception of the 1.1-ka event, we used the product method to define the earthquake times. The revised WS chronology yields a mean recurrence interval of 1.3 kyr (0.7–1.9-kyr estimated two-sigma [2δ] range based on interevent recurrence). These data help clarify the paleoearthquake history of the WS, including the important question of the timing and rupture extent of the most recent earthquake, and are essential to the improvement of earthquake-probability assessments for the Wasatch Front region.

  15. The effect of murine cytomegalovirus IE-3 specific shRNA is dependent on intragenic target site due to multiple transcription initiation sites

    PubMed Central

    2011-01-01

    Background Murine cytomegalovirus (MCMV) is closely related to human cytomegalovirus (HCMV) which is responsible for a variety of diseases, including retinitis, in immunocompromised individuals. Small inhibitory RNA molecules directed against essential viral regulatory genes may prove clinically useful. Methods Small hairpin RNAs (shRNAs) directed against the essential MCMV immediate early-3 gene (IE-3) were designed and tested in vitro at m.o.i.'s of 2 and 0.2 to determine if virus replication could be inhibited. Results At m.o.i. = 2, a MCMV IE-3 specific shRNA specific for sequences at the beginning of exon 5 inhibited virus replication with a maximum decrease in virus titer of approximately two logs at day 5 p.i. Surprisingly, however, at m.o.i. = 0.2, the same shRNA enhanced virus replication. In the latter case, the main IE-3 product observed in infected cells was not the expected 88 kd full length IE-3 protein observed at high m.o.i. but rather a truncated 45 kd form of this protein. Rapid analysis of 5' cDNA ends (5' RACE) indicated that substantial differences exist in the transcript profile produced by the IE-3 gene at low and high m.o.i. early after infection and that multiple transcripts are produced under both conditions. One such transcript, which originated in exon 5 of the IE-3 gene, was located outside the region targeted by our shRNA and was the major transcript produced at low m.o.i. Targeting of this exon 5 transcript with a second shRNA resulted in inhibition of virus replication at both low and high m.o.i. Conclusions These studies indicate that IE-3 has a complex transcriptional profile and that shRNA targeting of this and other viral regulatory genes which produce multiple transcripts may have unexpected effects on virus replication. PMID:21923934

  16. The effect of murine cytomegalovirus IE-3 specific shRNA is dependent on intragenic target site due to multiple transcription initiation sites.

    PubMed

    Marshall, Brendan; Zhang, Ming; Atherton, Sally S

    2011-09-18

    Murine cytomegalovirus (MCMV) is closely related to human cytomegalovirus (HCMV) which is responsible for a variety of diseases, including retinitis, in immunocompromised individuals. Small inhibitory RNA molecules directed against essential viral regulatory genes may prove clinically useful. Small hairpin RNAs (shRNAs) directed against the essential MCMV immediate early-3 gene (IE-3) were designed and tested in vitro at m.o.i.'s of 2 and 0.2 to determine if virus replication could be inhibited. At m.o.i. = 2, a MCMV IE-3 specific shRNA specific for sequences at the beginning of exon 5 inhibited virus replication with a maximum decrease in virus titer of approximately two logs at day 5 p.i. Surprisingly, however, at m.o.i. = 0.2, the same shRNA enhanced virus replication. In the latter case, the main IE-3 product observed in infected cells was not the expected 88 kd full length IE-3 protein observed at high m.o.i. but rather a truncated 45 kd form of this protein. Rapid analysis of 5' cDNA ends (5' RACE) indicated that substantial differences exist in the transcript profile produced by the IE-3 gene at low and high m.o.i. early after infection and that multiple transcripts are produced under both conditions. One such transcript, which originated in exon 5 of the IE-3 gene, was located outside the region targeted by our shRNA and was the major transcript produced at low m.o.i. Targeting of this exon 5 transcript with a second shRNA resulted in inhibition of virus replication at both low and high m.o.i. These studies indicate that IE-3 has a complex transcriptional profile and that shRNA targeting of this and other viral regulatory genes which produce multiple transcripts may have unexpected effects on virus replication.

  17. The Impact of Multiple-Site Interactions on the Energy Resolution of a High-Pressure Xenon Gamma-Ray Spectrometer

    SciTech Connect

    Kiff, Scott D.; He, Zhong

    2007-11-30

    High-pressure xenon (HPXe) ionization chambers have generated interest as a radiation detection medium for purposes requiring good energy resolution, high detection efficiency, and uniform response over a broad temperature range, such as homeland security and well logging applications. These chambers generally exhibit a substantial degradation of the measured energy resolution relative to theoretical limits. This investigation studies the impact of the number of interaction sites in an event sequence on the measured energy resolution using a benchmarked simulation package. The prominence of single and multiple-site interactions is investigated in addition to the photopeak broadening due to each event class. A radial position-sensing technique developed for coplanar-anode HPXe chambers is shown to have benefit for only single-site events.

  18. Rhodopsin TM6 Can Interact with Two Separate and Distinct Sites on Arrestin: Evidence for Structural Plasticity and Multiple Docking Modes in Arrestin–Rhodopsin Binding

    PubMed Central

    2015-01-01

    Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called “finger” loop (residues 67–79) and the other in the 160 loop (residues 155–165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin–rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin. PMID:24724832

  19. Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb, a Key Enzyme in Amylopectin Biosynthesis

    PubMed Central

    Makhmoudova, Amina; Williams, Declan; Brewer, Dyanne; Massey, Sarah; Patterson, Jenelle; Silva, Anjali; Vassall, Kenrick A.; Liu, Fushan; Subedi, Sanjeena; Harauz, George; Siu, K. W. Michael; Tetlow, Ian J.; Emes, Michael J.

    2014-01-01

    Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser649, Ser286, and Ser297. Two Ca2+-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser649 and Ser286 phosphorylation, and K2, responsible for Ser649 and Ser297 phosphorylation. The Ser286 and Ser297 phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser297 forms a stable salt bridge with Arg665, part of a conserved Cys-containing domain in plant branching enzymes. Ser649 conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application. PMID:24550386

  20. Identification of multiple phosphorylation sites on maize endosperm starch branching enzyme IIb, a key enzyme in amylopectin biosynthesis.

    PubMed

    Makhmoudova, Amina; Williams, Declan; Brewer, Dyanne; Massey, Sarah; Patterson, Jenelle; Silva, Anjali; Vassall, Kenrick A; Liu, Fushan; Subedi, Sanjeena; Harauz, George; Siu, K W Michael; Tetlow, Ian J; Emes, Michael J

    2014-03-28

    Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.

  1. Exposure to bleached kraft pulp mill effluent disrupts the pituitary-gonadal axis of white sucker at multiple sites

    SciTech Connect

    Van Der Kraak, G.J.; Munkittrick, K.R.; McMaster, M.E.; Portt, C.B.; Chang, J.P. )

    1992-08-01

    Recent studies have demonstrated reproductive problems in white sucker (Catostomus commersoni) exposed to bleached kraft pulp mill effluent (BKME) at Jackfish Bay on Lake Superior. These fish exhibit delayed sexual maturity, reduced gonadal size, reduced secondary sexual characteristics, and circulating steroid levels depressed relative to those of reference populations. The present studies were designed to evaluate sites in the pituitary-gonadal axis of prespawning white sucker affected by BKME exposure. At the time of entry to the spawning stream, plasma levels of immunoreactive gonadotropin (GtH)-II (LH-type GtH) in male and female white sucker were 30- and 50-fold lower, respectively, than the levels in fish from a reference site. A single intraperitoneal injection of D-Arg6, Pro9N-Et sGnRH (sGnRH-A, 0.1 mg/kg) increased plasma GtH levels in male and female fish at both sites, although the magnitude of the response was greatly reduced in BKME-exposed fish. Fish at the BKME site did not ovulate in response to sGnRH-A, while 10 of 10 fish from the reference site ovulated within 6 hr. Plasma 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) levels were depressed in BKME-exposed fish and unlike fish at the reference site, failed to increase in response to sGnRH-A. Testosterone levels in both sexes and 11-ketostestosterone levels in males were elevated in fish from the reference site but were not further increased by GnRH treatment. In contrast, BKME-exposed fish exhibit a transitory increase in testosterone levels in response to the GnRH analog. In vitro incubations of ovarian follicles obtained from fish at the BKME site revealed depressed basal secretion of testosterone and 17,20 beta-P and reduced responsiveness to the GtH analog human chorionic gonadotropin and to forskolin, a direct activator of adenylate cyclase.

  2. Optimising geological storage of CO2 by development of multiple injection sites in regionally extensive storage sandstones

    NASA Astrophysics Data System (ADS)

    Akhurst, Maxine; McDermott, Christopher; Williams, John; Mackay, Eric; Jin, Min; Tucker, Owain; Mallows, Tom; Hannis, Sarah; Pearce, Jonathan

    2016-04-01

    Carbon capture, transport and storage (CCS) is considered a key technology to provide secure, low-carbon energy supply and industrial processes to reduce the greenhouse gas emissions that contribute to the adverse effects of climatic change. Geological storage of carbon dioxide (CO2), captured during hydrocarbon production at the Sleipner Field, in strata beneath the Norwegian sector of the North Sea has been in operation since 1996. Projects to store CO2 captured at power plants in strata underlying the North Sea are currently in design. Storage of the CO2 is planned in depleted hydrocarbon fields or regionally extensive sandstones containing brine (saline aquifer sandstones). The vast majority of the UK potential storage resource is within brine-saturated sandstone formations. The sandstone formations are each hundreds to thousands of square kilometres in extent and underlie all sectors of the North Sea. The immense potential to store CO2 in these rocks can only be fully achieved by the operation of more than one injection site within each formation. Here we report an investigation into the operation of more than one injection site within a storage formation using a UK North Sea case study of the Captain Sandstone and the included Goldeneye Field, which is part of the mature hydrocarbon province offshore Scotland. Research by the CO2MultiStore project was targeted to increase understanding and confidence in the operation of two sites within the Captain Sandstone. Methods were implemented to reduce the effort and resources needed to characterise the sandstone, and increase understanding of its stability and performance during operation of more than one injection site. Generic learning was captured throughout the research relevant to the characterisation of extensive storage sandstones, management of the planned injection operations and monitoring of CO2 injection at two (or more) sites within any connected sandstone formation. The storage of CO2 can be optimised

  3. 15 CFR 921.33 - Boundary changes, amendments to the management plan, and addition of multiple-site components.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Reserve, may be made only after written approval by NOAA. NOAA may require public notice, including notice... statement may be required. NOAA will place a notice in the Federal Register of any proposed changes in... made. NOAA will publish notice of the proposed new site including an invitation for comments from...

  4. Influence of the site of arterial occlusion on multiple baseline hemodynamic MRI parameters and post-thrombolytic recanalization in acute stroke.

    PubMed

    Derex, L; Hermier, M; Adeleine, P; Pialat, J B; Wiart, M; Berthezène, Y; Froment, J C; Trouillas, P; Nighoghossian, N

    2004-11-01

    In this prospective MRI study, we evaluated the impact of the site of occlusion on multiple baseline perfusion parameters and subsequent recanalization in 49 stroke patients who were given intravenous tissue plasminogen activator (tPA). Pretreatment magnetic resonance angiography (MRA) revealed an arterial occlusion in 47 patients: (1) internal carotid artery (ICA) + M1 middle cerebral artery (MCA) occlusion (n=12); (2) M1 MCA occlusion (n=19); (3) M2 MCA, distal branches of the MCA and anterior cerebral artery (ACA) occlusion (n=16). Patients with ICA occlusion had significantly larger DWI, PWI and mismatch lesion volume on pretreatment MRI compared to patients with other sites of occlusion. The differences in cerebral blood flow (CBF) and peak height were significantly higher in patients with ICA occlusion compared to patients with other sites of occlusion (P=0.03 and P=0.04, respectively). Day 1 MRA showed recanalization in 28 patients (60%). The rate of recanalization was significantly different depending on the site of occlusion: 33% in ICA + M1 MCA occlusion, 63% in M1 MCA occlusion and 81% in either M2 MCA, distal branches of the MCA or ACA occlusion (P=0.002). Our data suggest that CBF and peak height are the most relevant MRI parameters to assess the severity of hemodynamic impairment in regard to the site of occlusion.

  5. A G-to-T transversion at the splice acceptor site of dystrophin exon 14 shows multiple splicing outcomes that are not exemplified by transition mutations.

    PubMed

    Ota, Mitsunori; Takeshima, Yasuhiro; Nishida, Atsushi; Awano, Hiroyuki; Lee, Tomoko; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    Mutations at splicing consensus sequences have been shown to induce splicing errors such as exon skipping or cryptic splice site activation. Here, we identified eight splicing products caused by a G-to-T transversion mutation at the splice acceptor site of exon 14 of the dystrophin gene (c.1603-1G>T). Unexpectedly, the most abundant product showed skipping of the two consecutive exons 14 and 15, and exon 14 skipping was observed as the second most abundant product. To examine the cause of this splicing multiplicity, minigenes containing dystrophin exons 14 and 15 with their flanking introns were constructed and subjected to in vitro splicing. Minigenes with the wild-type sequence or a G>A transition at position c.1603-1 produced only the mature mRNA. On the other hand, the minigenes with a G>T or G>C transversion mutation produced multiple splicing products. A time-course analysis of the in vitro splicing revealed that splicing of the middle intron, intron 14, was the first step in transcript maturation for all four minigene constructs. The identity of the mutant nucleotide, but not its position, is a factor leading to multiple splicing outcomes. Our results suggest that exon skipping therapy for Duchenne's muscular dystrophy should be carefully monitored for their splicing outcomes.

  6. Interim guidelines for protecting fire-fighting personnel from multiple hazards at nuclear plant sites: Final report

    SciTech Connect

    Klein, A.R.; Bloom, C.W.

    1989-07-01

    This report provides interim guidelines for reducing the impact to fire fighting and other supporting emergency response personnel from the multiple hazards of radiation, heat stress, and trauma when fighting a fire in a United States commercial nuclear power plant. Interim guidelines are provided for fire brigade composition, training, equipment, procedures, strategies, heat stress and trauma. In addition, task definitions are provided to evaluate and further enhance the interim guidelines over the long term. 19 refs.

  7. Correcting for Incomplete Saturation and Off-Resonance Effects in Multiple-Site Saturation-Transfer Kinetic Measurements

    NASA Astrophysics Data System (ADS)

    Kingsley, Peter B.; Monahan, W. Gordon

    2000-09-01

    The effects of incomplete saturation and off-resonance irradiation on nuclear magnetic resonance saturation-transfer measurements of three-site chemical-exchange rates are discussed. A new method that uses double-saturation measurements is compared with two published methods, one that uses single-saturation measurements and one that uses a single-saturation measurement and a double-saturation measurement. Several formulas are compared for measuring the exchange rate constant kDE for exchange from a detected spin D to an exchanging spin E in the presence of exchange from spin D to a competing spin C. For each method, formulas are derived with corrections for incomplete saturation or off-resonance effects, with both corrections, and with neither correction. Exact formulas are available for three exchanging sites with incomplete saturation if there are no off-resonance effects. Off-resonance corrections are imperfect even with complete saturation.

  8. Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for GA-binding protein (GABP)

    PubMed Central

    Kumar, Premlata; Ward, Bryan K.; Minchin, Rodney F.; Ratajczak, Thomas

    2001-01-01

    Within steroid receptor heterocomplexes the large tetratricopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (Hsp90) and act coordinately with Hsp90 to modulate receptor activity. The reversible nature of the interaction between the immunophilins and Hsp90 suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a 5-kilobase (kb) 5′-flanking region of the human gene and demonstrated that a ∼50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABPα and GABPβ subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GABP is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression. PMID:11525247

  9. Multiple actions of glaucine on cyclic nucleotide phosphodiesterases, alpha 1-adrenoceptor and benzothiazepine binding site at the calcium channel.

    PubMed Central

    Ivorra, M. D.; Lugnier, C.; Schott, C.; Catret, M.; Noguera, M. A.; Anselmi, E.; D'Ocon, P.

    1992-01-01

    1. In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of alpha-adrenoceptors, by use of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. Contraction evoked by noradrenaline (1 microM) or depolarizing solution (60 mM KCl) were inhibited in a concentration-dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA-induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl-induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically. 3. In Ca(2+)-free solution, prazosin (0.1 microM) and glaucine (0.1 mM) inhibited the contraction evoked by NA; diltiazem (0.1 mM) diminished this contraction whereas nifedipine (1 microM) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mM) significantly inhibited the contractions evoked by NA or caffeine in Ca(2+)-free medium. 4. Glaucine and papaverine show affinity at the [3H]-prazosin binding site and at the benzothiazepine binding site of the Ca(2+)-channel receptor complex, but have no effect at the dihydropyridine binding site in rat cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1327380

  10. Gymnotus coatesi (Gymnotiformes): A Case of Colocation of Multiple Sites of 18S rDNA with Telomeric Sequences.

    PubMed

    Machado, Milla de Andrade; Cardoso, Adauto Lima; Milhomem-Paixão, Susana Suely Rodrigues; Pieczarka, Julio Cesar; Nagamachi, Cleusa Yoshiko

    2017-10-01

    Gymnotus coatesi is a small and rare species of banded knife fish that was originally described by LaMonte in 1935, found along the main stretch of the Amazon River. There is no described cytogenetic data on this species. We analyzed the karyotype of five specimens of G. coatesi collected from Cururutuia Stream in Bragança, Pará, Brazil. The obtained diploid number is 50 and the karyotypic formula is 24 m/sm +26 st/a. The constitutive heterochromatin is DAPI positive and distributed mainly in the centromeric and pericentromeric regions of the chromosomes. Ag-nucleolus organizer regions staining showed nine active sites. The 5S rDNA probe hybridized chromosome pair 17 in the interstitial part of the long arm. Fluorescence in situ hybridization (FISH) with telomeric probes revealed signals only at terminal regions of the chromosomes. The 18S rDNA probe hybridized to 21 sites, and these signals colocalized with the telomeric sequences. This relatively high number of 18S rDNA sites may reflect gene duplication mediated by transposable elements. These results indicate that although the diploid number of G. coatesi is within the range previously observed for other members of the genus, various karyotypic characteristics distinguish G. coatesi from the other species of the genus and members of the Gymnotiform order.

  11. JET2 Viewer: a database of predicted multiple, possibly overlapping, protein–protein interaction sites for PDB structures

    PubMed Central

    Ripoche, Hugues; Laine, Elodie; Ceres, Nicoletta; Carbone, Alessandra

    2017-01-01

    The database JET2 Viewer, openly accessible at http://www.jet2viewer.upmc.fr/, reports putative protein binding sites for all three-dimensional (3D) structures available in the Protein Data Bank (PDB). This knowledge base was generated by applying the computational method JET2 at large-scale on more than 20 000 chains. JET2 strategy yields very precise predictions of interacting surfaces and unravels their evolutionary process and complexity. JET2 Viewer provides an online intelligent display, including interactive 3D visualization of the binding sites mapped onto PDB structures and suitable files recording JET2 analyses. Predictions were evaluated on more than 15 000 experimentally characterized protein interfaces. This is, to our knowledge, the largest evaluation of a protein binding site prediction method. The overall performance of JET2 on all interfaces are: Sen = 52.52, PPV = 51.24, Spe = 80.05, Acc = 75.89. The data can be used to foster new strategies for protein–protein interactions modulation and interaction surface redesign. PMID:27899675

  12. DEVELOPING SITE-SPECIFIC DERIVED CONCENTRATION GUIDELINE LEVELS FOR MULTIPLE MEDIA AT THE CONNECTICUT YANKEE HADDAM NECK PLANT

    SciTech Connect

    Taylor, S.W.; Smith, L.C.; Carr, R.K.; Carson, A.; Darois, E.

    2003-02-27

    As part of the license termination process, site-specific Derived Concentration Guideline Levels for the Haddam Neck Plant site are developed for soil, groundwater, concrete left standing, and concrete demolished that satisfy the radiological criteria for unrestricted use as defined in 10 CFR 20.1402. Background information on the license termination process and characteristics of the Haddam Neck Plant site are presented. The dose models and associated resident farmer and building occupancy scenarios, applicable pathways, and critical groups developed to establish the Derived Concentration Guideline Levels are described. A parameter assignment process is introduced wherein general population values are used to establish behavioral and metabolic parameters representative of an average member of the critical group, while the uncertainty associated with important physical parameters is considered. A key element of the parameter assignment process is the use of sensitivity analysis to identify the dose sensitive physical parameters and to ensure that such parameters are assigned conservative values. Structuring the parameter assignment process, completing the formal sensitivity analyses, and assigning conservative values to the sensitive physical parameters in a consistent way establishes a calculation framework that lead to Derived Concentration Guideline Levels with a uniform level of conservatism across all media and all radionuclides.

  13. The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria.

    PubMed

    Sung, Tzu-Ying; Tseng, Ching-Chih; Hsieh, Ming-Hsiun

    2010-08-01

    In Arabidopsis, RNA editing changes more than 500 cytidines to uridines in mitochondrial transcripts. The editing enzyme and co-factors involved in these processes are largely unknown. We have identified a nuclear gene SLOW GROWTH1 (SLO1) encoding an E motif-containing pentatricopeptide repeat protein that is required for RNA editing of nad4 and nad9 in Arabidopsis mitochondria. The SLO1 protein is localized to the mitochondrion, and its absence gives rise to small plants with slow growth and delayed development. A survey of approximately 500 mitochondrial RNA editing sites in Arabidopsis reveals that the editing of two sites, nad4-449 and nad9-328, is abolished in the slo1 mutants. Sequence comparison in the upstream (from -1 to -15 bp) of nad4-449 and nad9-328 editing sites shows that nine of the 15 nucleotides are identical. In addition to RNA editing, we used RNA gel blot analysis to compare the abundance and banding patterns of mitochondrial transcripts between the wild type and slo1 mutants. Of the 79 genes and open reading frames examined, steady-state levels of 56 mitochondrial transcripts are increased in the slo1 mutants. These results suggest that the SLO1 protein may indirectly regulate plant growth and development via affecting mitochondrial RNA editing and gene expression.

  14. Analysis of quadrupole splitting of multiple Fe sites intermixed in Si(111) with Mössbauer spectroscopy

    NASA Astrophysics Data System (ADS)

    Kawauchi, Taizo; Zhang, Xiaowei; Fukutani, Katsuyuki

    2016-12-01

    The iron silicide has various interesting phases both fundamentally and technologically, which have acquired much attention to date. Iron silicides are often fabricated on a Si substrate by a solid phase epitaxy method, and the initial stage of intermixing of iron atoms with substrate Si is of crucial importance for silicide fabrication, which remains to be clarified. Here, we have investigated the initial stage of the iron-silicide formation before crystallization with Mössbauer spectroscopy suited to characterization of magnetic and chemical properties of 57Fe atoms in materials. The sample was prepared by deposition of 57Fe of 1 nm on a Si(111) surface at 450 K. Conventional Mössbauer spectroscopy in the energy domain revealed presence of two iron sites with similar quadrupole splits and isomer shifts, which hampered complete analysis of this system. By combining the time-domain spectroscopy using polarized synchrotron radiation, we have separately analyzed the quadrupole splits and isomer shifts for the two iron sites. By using the theoretical simulation, furthermore, we successfully reproduced the experimentally observed time spectrum of the nuclear resonant scattering on the assumption that iron atoms randomly occupy the substitutional sites for Si at the initial stage of intermixing before crystallization of an iron silicide.

  15. Closure Strategy for a Waste Disposal Facility with Multiple Waste Types and Regulatory Drivers at the Nevada Test Site

    SciTech Connect

    D. Wieland, V. Yucel, L. Desotell, G. Shott, J. Wrapp

    2008-04-01

    The U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) plans to close the waste and classified material storage cells in the southeast quadrant of the Area 5 Radioactive Waste Management Site (RWMS), informally known as the '92-Acre Area', by 2011. The 25 shallow trenches and pits and the 13 Greater Confinement Disposal (GCD) borings contain various waste streams including low-level waste (LLW), low-level mixed waste (LLMW), transuranic (TRU), mixed transuranic (MTRU), and high specific activity LLW. The cells are managed under several regulatory and permit programs by the U.S. Department of Energy (DOE) and the Nevada Division of Environmental Protection (NDEP). Although the specific closure requirements for each cell vary, 37 closely spaced cells will be closed under a single integrated monolayer evapotranspirative (ET) final cover. One cell will be closed under a separate cover concurrently. The site setting and climate constrain transport pathways and are factors in the technical approach to closure and performance assessment. Successful implementation of the integrated closure plan requires excellent communication and coordination between NNSA/NSO and the regulators.

  16. Modeling Above-Ground Biomass Across Multiple Circum-Arctic Tundra Sites Using High Spatial Resolution Remote Sensing

    NASA Astrophysics Data System (ADS)

    Räsänen, Aleksi; Juutinen, Sari; Aurela, Mika; Virtanen, Tarmo

    2017-04-01

    Biomass is one of the central bio-geophysical variables in Earth observation for tracking plant productivity, and flow of carbon, nutrients, and water. Most of the satellite based biomass mapping exercises in Arctic environments have been performed by using rather coarse spatial resolution data, e.g. Landsat and AVHRR which have spatial resolutions of 30 m and >1 km, respectively. While the coarse resolution images have high temporal resolution, they are incapable of capturing the fragmented nature of tundra environment and fine-scale changes in vegetation and carbon exchange patterns. Very high spatial resolution (VHSR, spatial resolution 0.5-2 m) satellite images have the potential to detect environmental variables with an ecologically sound spatial resolution. The usage of VHSR images has, nevertheless, been modest so far in biomass modeling in the Arctic. Our objectives were to use VHSR for predicting above ground biomass in tundra landscapes, evaluate whether a common predictive model can be applied across circum-Arctic tundra and peatland sites having different types of vegetation, and produce knowledge on distribution of plant functional types (PFT) in these sites. Such model development is dependent on ground-based surveys of vegetation with the same spatial resolution and extent with the VHSR images. In this study, we conducted ground-based surveys of vegetation composition and biomass in four different arctic tundra or peatland areas located in Russia, Canada, and Finland. First, we sorted species into PFTs and developed PFT-specific models to predict biomass on the basis of non-destructive measurements (cover, height). Second, we predicted overall biomass on landscape scale by combinations of single bands and vegetation indices of very high resolution satellite images (QuickBird or WorldView-2 images of the eight sites). We compared area-specific empirical regression models and common models that were applied across all sites. We found that NDVI was

  17. Closure Strategy for a Waste Disposal Facility with Multiple Waste Types and Regulatory Drivers at the Nevada Test Site

    SciTech Connect

    L. Desotell; D. Wieland; V. Yucel; G. Shott; J. Wrapp

    2008-03-01

    The U.S. Department of Energy, National Security Administration Nevada Site Office (NNSA/NSO) is planning to close the 92-Acre Area of the Area 5 Radioactive Waste Management Site (RWMS) at the Nevada Test Site (NTS), which is about 65 miles northwest of Las Vegas, Nevada. Closure planning for this facility must take into account the regulatory requirements for a diversity of waste streams, disposal and storage configurations, disposal history, and site conditions. This paper provides a brief background of the Area 5 RWMS, identifies key closure issues, and presents the closure strategy. Disposals have been made in 25 shallow excavated pits and trenches and 13 Greater Confinement Disposal (GCD) boreholes at the 92-Acre Area since 1961. The pits and trenches have been used to dispose unclassified low-level waste (LLW), low-level mixed waste (LLMW), and asbestiform waste, and to store classified low-level and low-level mixed materials. The GCD boreholes are intermediate-depth disposal units about 10 feet (ft) in diameter and 120 ft deep. Classified and unclassified high-specific activity LLW, transuranic (TRU), and mixed TRU are disposed in the GCD boreholes. TRU waste was also disposed inadvertently in trench T-04C. Except for three disposal units that are active, all pits and trenches are operationally covered with 8-ft thick alluvium. The 92-Acre Area also includes a Mixed Waste Disposal Unit (MWDU) operating under Resource Conservation and Recovery Act (RCRA) Interim Status, and an asbestiform waste unit operating under a state of Nevada Solid Waste Disposal Site Permit. A single final closure cover is envisioned over the 92-Acre Area. The cover is the evapotranspirative-type cover that has been successfully employed at the NTS. Closure, post-closure care, and monitoring must meet the requirements of the following regulations: U.S. Department of Energy Order 435.1, Title 40 Code of Federal Regulations (CFR) Part 191, Title 40 CFR Part 265, Nevada Administrative

  18. Ferromanganese nodules from MANOP Sites H, S, and R-Control of mineralogical and chemical composition by multiple accretionary processes

    USGS Publications Warehouse

    Dymond, J.; Lyle, M.; Finney, B.; Piper, D.Z.; Murphy, K.; Conard, R.; Pisias, N.

    1984-01-01

    The chemical composition of ferromanganese nodules from the three nodule-bearing MANOP sites in the Pacific can be accounted for in a qualitative way by variable contributions of distinct accretionary processes. These accretionary modes are: 1. (1) hydrogenous, i.e., direct precipitation or accumulation of colloidal metal oxides in seawater, 2. (2) oxic diagenesis which refers to a variety of ferromanganese accretion processes occurring in oxic sediments; and 3. (3) suboxic diagenesis which results from reduction of Mn+4 by oxidation of organic matter in the sediments. Geochemical evidence suggests processes (1) and (2) occur at all three MANOP nodule-bearing sites, and process (3) occurs only at the hemipelagic site, H, which underlies the relatively productive waters of the eastern tropical Pacific. A normative model quantitatively accounts for the variability observed in nearly all elements. Zn and Na, however, are not well explained by the three end-member model, and we suggest that an additional accretionary process results in greater variability in the abundances of these elements. Variable contributions from the three accretionary processes result in distinct top-bottom compositional differences at the three sites. Nodule tops from H are enriched in Ni, Cu, and Zn, instead of the more typical enrichments of these elements in nodule bottoms. In addition, elemental correlations typical of most pelagic nodules are reversed at site H. The three accretionary processes result in distinct mineralogies. Hydrogenous precipitation produces ??MnO2. Oxic diagenesis, however, produces Cu-Ni-rich todorokite, and suboxic diagenesis results in an unstable todorokite which transforms to a 7 A?? phase ("birnessite") upon dehydration. The presence of Cu and Ni as charge-balancing cations influence the stability of the todorokite structure. In the bottoms of H nodules, which accrete dominantly by suboxic diagenesis, Na+ and possibly Mn+2 provide much of the charge balance for

  19. Using multiple indices to evaluate scenarios for the remediation of contaminated land: the Porto Marghera (Venice, Italy) contaminated site.

    PubMed

    Critto, Andrea; Agostini, Paola

    2009-09-01

    The management of contaminated sites requires the investigation of different involved aspects (from socioeconomic to risk and technological issues) and the presentation of useful and condensed information to decision makers. For this purpose, indices are more and more recognized as effective and valuable tools. This paper presents specific indices created within the DEcision Support sYstem for REhabilitation of contaminated sites (DESYRE). DESYRE is a software which aids decision making for the rehabilitation of a large contaminated site (i.e., megasite) by the creation and comparison of different rehabilitation alternatives. The software is composed of six modules, each dealing with a specific aspect of the remediation process, ending with the decision module. In this module, scenarios (i.e., suitable solutions for the rehabilitation of the contaminated site including selected land use, socioeconomic benefits, remediation costs, time span, environmental impacts, technology set/s, and residual risk) are created and evaluated by means of suitable indices. Nine indices cover the socioeconomic, risk, technological, cost, time, and environmental impact aspects. Mathematical algorithms are used to calculate these indices by taking into account data collected during the analytical steps of the DESYRE system and elaborated through the support of the spatial analysis, which is embedded in the system. The case study of Porto Marghera, Venice, Italy is presented in order to document the effectiveness of developed indices in evaluating management solutions and presenting options to decision makers. For the purpose of this study, three different scenarios for the remediation of a part of the site of Porto Marghera (approximately 530 ha) are developed and compared. The three scenarios consider the industrial land use and deal with the contamination in soil caused by inorganic and organic compounds. The scenarios mainly differ for the number of the included remediation

  20. Evaluating multiple indices of agricultural water use efficiency and productivity to improve comparisons between sites and trends

    NASA Astrophysics Data System (ADS)

    Levy, M. C.

    2012-12-01

    Approximately 70% of global available freshwater supplies are used in the agricultural sector. Increased demands for water to meet growing population food requirements, and expected changes in the reliability of freshwater supplies due to climate change, threaten the sustainability of water supplies worldwide - not only on farms, but in connected cities and industries. Researchers concerned with agricultural water use sustainability use a variety of theoretical and empirical measures of efficiency and productivity to gain insight into the sustainability of agricultural water use. However, definitions of measures, or indices, vary between different natural and political boundaries, across regions, states and nations and between their respective research, industry, and environmental groups. Index development responds to local data availability and local agendas, and there is debate about the validity of various indices. However, real differences in empirical index measures are not well-understood across the multiple disciplines that study agricultural water use, including engineering and hydrology, agronomy, climate and soil sciences, and economics. Nevertheless reliable, accessible, and generalizable indices are required for planners and policymakers to promote sustainable water use systems. This study synthesizes a set of water use efficiency and productivity indices based on academic, industry and government literature in California and Australia, two locations with similarly water-stressed and valuable agricultural industries under pressure to achieve optimal water use efficiency and productivity. Empirical data at the irrigation district level from the California San Joaquin Valley and Murray Darling Basin states of Victoria and New South Wales in Australia are used to compute indices that estimate efficiency, yield productivity, and economic productivity of agricultural water use. Multiple index estimates of same time-series data demonstrate historical spread

  1. Metachronous multiple carcinomas arising at a colostomy site 15 years after abdominoperineal resection: a case report and review of Japanese literature.

    PubMed

    Okamoto, Norihiro; Maeda, Koutarou; Hanai, Tsunekazu; Sato, Harunobu; Masumori, Kouji; Katsuno, Hidetoshi; Maruta, Morito

    2009-01-01

    Metachronous multiple carcinomas at the site of a stoma is a rare condition after surgery. A 67-year-old man with a second tumor at the stoma site 15 years after abdominoperineal resection for rectal carcinoma is reported herein with a review of the Japanese literature. The patient visited our hospital 20 years after initial surgery, presenting with constipation and increasing tumor size in the stoma. A biopsy specimen of the tumor revealed adenocarcinoma. Block resection of the sigmoid colon and colostomy with the adjacent abdominal wall was performed. Histopathological examination showed a well-differentiated adenocarcinoma in the stoma invading the adjacent skin. A median of 144 months from the initial operation was documented in 23 cases reported.

  2. Interpretation of Ocular Melanin Drug Binding Assays. Alternatives to the Model of Multiple Classes of Independent Sites.

    PubMed

    Manzanares, José A; Rimpelä, Anna-Kaisa; Urtti, Arto

    2016-04-04

    Melanin has a high binding affinity for a wide range of drugs. The determination of the melanin binding capacity and its binding affinity are important, e.g., in the determination of the ocular drug distribution, the prediction of drug effects in the eye, and the trans-scleral drug delivery. The binding parameters estimated from a given data set vary significantly when using different isotherms or different nonlinear fitting methods. In this work, the commonly used bi-Langmuir isotherm, which assumes two classes of independent sites, is confronted with the Sips isotherm. Direct, log-log, and Scatchard plots are used, and the interpretation of the binding curves in the latter is critically analyzed. In addition to the goodness of fit, the emphasis is placed on the physical meaning of the binding parameters. The bi-Langmuir model imposes a bimodal distribution of binding energies for the sites on the melanin granules, but the actual distribution is most likely continuous and unimodal, as assumed by the Sips isotherm. Hence, the latter describes more accurately the distribution of binding energies and also the experimental results of melanin binding to drugs and metal ions. Simulations are used to show that the existence of two classes of sites cannot be confirmed on the sole basis of the shape of the binding curve in the Scatchard plot, and that serious doubts may appear on the meaning of the binding parameters of the bi-Langmuir model. Experimental results of melanin binding to chloroquine and metoprolol are used to illustrate the importance of the choice of the binding isotherm and of the method used to evaluate the binding parameters.

  3. Acute Haemodynamic and Echocardiographic Effects of Multiple Configurations of Left Ventricular Pacing Sites in Acute Myocardial Infarction: Experimental Study.

    PubMed

    Matthaios, I; Kaladaridou, A; Skaltsiotes, E; Agrios, J; Antoniou, A; Georgiopoulos, G; Papadopoulou, E; Pamboucas, C; Toumanidis, S

    2017-04-01

    Left ventricular (LV) pacing is unsuccessful in a significant number of patients, mainly due to sub-optimal LV pacing location. Nevertheless, data about the impact of different pacing sites on LV function in ischaemic myocardium are scarce. The purpose of this study was to investigate the effect of combinations of alternative LV pacing sites on LV mechanics after experimental acute anterior myocardial infarction (AMI), in order to define the optimal configuration. Atrioventricular epicardial pacing at alternative pacing sites was performed in 16 healthy pigs simultaneously, after experimental AMI. Standard right ventricular (RV) apical pacing was combined with: i) LV apex lateral wall; ii) LV basal posterior wall; iii) LV basal anterior wall, and; iv) LV basal anterior wall + LV basal posterior wall. Moreover the pacing configurations of, v) LV basal posterior wall + LV apex lateral wall; vi) LV basal posterior wall + LV basal anterior wall, and; vii) LV basal anterior wall + LV apex lateral wall were also investigated. Haemodynamic parameters, together with classic and novel echocardiographic indices were used, to evaluate the effect of each pacing combination. A speckle tracking technique using EchoPAC software was used. After AMI, the pacing combination of LV apex lateral wall and LV basal posterior wall had the most favourable effect on LV function, leading to similar haemodynamic and torsional effects with sinus rhythm (all variables p>0.05). In pig hearts after AMI, the combination of pacing LV apex lateral wall and LV basal posterior wall managed to maintain the LV function at a level comparable to the sinus rhythm. Copyright © 2016 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.

  4. Constraining the factor analytical solutions obtained from multiple-year receptor modeling of ambient PM2.5 data from five speciation sites in Ontario, Canada

    NASA Astrophysics Data System (ADS)

    Sofowote, Uwayemi M.; Su, Yushan; Dabek-Zlotorzynska, Ewa; Rastogi, Ankit K.; Brook, Jeff; Hopke, Philip K.

    2015-05-01

    Rotational ambiguity in factor analyses leads to solutions that are not always consistent with reality. The inherent non-negativity constraints in positive matrix factorization (PMF) help to prevent factor solutions from becoming overly unrealistic, but they are not sufficient to prevent unwanted rotations that could manifest in factors that should have similar compositions varying across multiple sites. The Canadian National Air Pollution Surveillance (NAPS) network operates five fine particulate matter (PM2.5) speciation sites in Ontario. Data from these sites from 2005 to 2010 were subjected to PMF to obtain factors representing sources of particulate matter. Eight factors were found to be common across these sites. These factors had profiles that varied greatly from one site to the other, suggesting that the PMF solutions were impacted by some rotational ambiguity. New features in the EPA PMF V5 program allow the use of a priori information to impose mathematical constraints that guide the evolution of the factor solutions. These constraints reduce the rotational space. In situations where major emissions sources are known and located in the neighborhood of receptors, or emissions inventories and literature source profiles exist, it is easy to use these profiles to force the factor solutions to conform to the expected signatures. In our case, reported source profiles were neither available nor applicable due to the large spatial span of potential sources and receptor sites. This work describes how such constraints can be generated and used in these complex situations. The fundamental principle explored in this work is the concept of 'stiffness' of PMF solutions to identify the desirable non-rotating factors.

  5. Variance component analysis of a multi-site study for the reproducibility of multiple reaction monitoring measurements of peptides in human plasma.

    PubMed

    Xia, Jessie Q; Sedransk, Nell; Feng, Xingdong

    2011-01-26

    In the Addona et al. paper (Nature Biotechnology 2009), a large-scale multi-site study was performed to quantify Multiple Reaction Monitoring (MRM) measurements of proteins spiked in human plasma. The unlabeled signature peptides derived from the seven target proteins were measured at nine different concentration levels, and their isotopic counterparts were served as the internal standards. In this paper, the sources of variation are analyzed by decomposing the variance into parts attributable to specific experimental factors: technical replicates, sites, peptides, transitions within each peptide, and higher-order interaction terms based on carefully built mixed effects models. The factors of peptides and transitions are shown to be major contributors to the variance of the measurements considering heavy (isotopic) peptides alone. For the light ((12)C) peptides alone, in addition to these factors, the factor of study*peptide also contributes significantly to the variance of the measurements. Heterogeneous peptide component models as well as influence analysis identify the outlier peptides in the study, which are then excluded from the analysis. Using a log-log scale transformation and subtracting the heavy/isotopic peptide [internal standard] measurement from the peptide measurements (i.e., taking the logarithm of the peak area ratio in the original scale establishes that), the MRM measurements are overall consistent across laboratories following the same standard operating procedures, and the variance components related to sites, transitions and higher-order interaction terms involving sites have greatly reduced impact. Thus the heavy peptides have been effective in reducing apparent inter-site variability. In addition, the estimates of intercepts and slopes of the calibration curves are calculated for the sub-studies. The MRM measurements are overall consistent across laboratories following the same standard operating procedures, and heavy peptides can be used as

  6. Fibronectin at select sites binds multiple growth factors and enhances their activity: expansion of the collaborative ECM-GF paradigm.

    PubMed

    Zhu, Jia; Clark, Richard A F

    2014-04-01

    Intensive research has demonstrated that extracellular matrix (ECM) molecules and growth factors (GF) collaborate at many different levels. The ability of ECM to modulate GF signals has important implications in tissue formation and homeostasis as well as novel therapies for acute and chronic wounds. Recently, a number of GF-binding sites was identified in fibronectin (FN) and was shown to provide another layer of regulation on GF signaling. Here, we review these new findings on FN interaction with GF in the context of general ways ECM molecules regulate GF signaling.

  7. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms

    PubMed Central

    Leakey, Tatiana I.; Zielinski, Jerzy; Siegfried, Rachel N.; Siegel, Eric R.; Fan, Chun-Yang; Cooney, Craig A.

    2008-01-01

    DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites. PMID:18480118

  8. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms.

    PubMed

    Leakey, Tatiana I; Zielinski, Jerzy; Siegfried, Rachel N; Siegel, Eric R; Fan, Chun-Yang; Cooney, Craig A

    2008-06-01

    DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites.

  9. Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites

    PubMed Central

    Zhou, Jin; Tracy, Timothy S.

    2010-01-01

    UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration-dependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion. PMID:20007295

  10. Glucuronidation of dihydrotestosterone and trans-androsterone by recombinant UDP-glucuronosyltransferase (UGT) 1A4: evidence for multiple UGT1A4 aglycone binding sites.

    PubMed

    Zhou, Jin; Tracy, Timothy S; Remmel, Rory P

    2010-03-01

    UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration-dependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.

  11. Safety, immunogenicity and preliminary efficacy of multiple-site vaccination with an Epidermal Growth Factor (EGF) based cancer vaccine in advanced non small cell lung cancer (NSCLC) patients

    PubMed Central

    2011-01-01

    The prognosis of patients with advanced non small cell lung (NSCLC) cancer remains dismal. Epidermal Growth Factor Receptor is over-expressed in many epithelial derived tumors and its role in the development and progression of NSCLC is widely documented. CimaVax-EGF is a therapeutic cancer vaccine composed by human recombinant Epidermal Growth Factor (EGF) conjugated to a carrier protein, P64K from Neisseria Meningitides. The vaccine is intended to induce antibodies against self EGF that would block EGF-EGFR interaction. CimaVax-EGF has been evaluated so far in more than 1000 advanced NSCLC patients, as second line therapy. Two separate studies were compared to assess the impact of high dose vaccination at multiple anatomic sites in terms of immunogenicity, safety and preliminary efficacy in stage IIIb/IV NSCLC patients. In both clinical trials, patients started vaccination 1 month after finishing first line chemotherapy. Vaccination at 4 sites with 2.4 mg of EGF (high dose) was very safe. The most frequent adverse events were grade 1 or 2 injection site reactions, fever, headache and vomiting. Patients had a trend toward higher antibody response. The percent of very good responders significantly augmented and there was a faster decrease of circulating EGF. All vaccinated patients and those classified as good responders immunized with high dose at 4 sites, had a large tendency to improved survival. PMID:22024351

  12. Prevalence and concordance of human papillomavirus infection at multiple anatomic sites among HIV-infected women from Chennai, India.

    PubMed

    Menezes, Lynette J; Poongulali, Selvamuthu; Tommasino, Massimo; Lin, Hui-Yi; Kumarasamy, Nagalingeswaran; Fisher, Kate J; Saravanan, Shanmugam; Gheit, Tarik; Ezhilarasi, Chandrasekaran; Jeeva, Arumugham; Lu, Beibei; Giuliano, Anna R

    2016-06-01

    Human papillomavirus (HPV) infection at the cervix, anus and oropharynx has been rarely concurrently estimated among HIV-infected women. Using multiplex polymerase chain reaction testing, we prospectively evaluated HPV genotype distribution across three anatomic sites among 50 eligible HIV-infected women from Chennai, India, who provided biological specimens and answered a sexual behaviour questionnaire. We also assessed clinical and behavioural factors related to HPV prevalence. Oncogenic HPV prevalence was comparable between the anus and cervix at 52.2% and 52.0% and lower at the oropharynx at 13.2%; 78% of women with a cervical HPV infection had the same type in the anus. Newly acquired oncogenic HPV infections were lower at cervix (24%) than anus (35%) at three months. 'Any type' cervical HPV prevalence was higher among women with low education and less than five years since HIV diagnosis. CD4+ count and antiretroviral therapy status were not associated with HPV prevalence at the three anatomic sites; however, enrolment cervical HPV16 prevalence was elevated among women with nadir CD4+ <200 cells/µL and enrolment CD4+ <350 cells/µL. Regular cervical screening is essential in HIV-infected Indian women irrespective of CD4+ count and antiretroviral therapy status. Additional research clarifying the natural history of anal HPV infection is also needed in this population. © The Author(s) 2015.

  13. The red wine antioxidant resveratrol prevents cardiomyocyte injury following ischemia-reperfusion via multiple sites and mechanisms.

    PubMed

    Goh, Siew Simg C; Woodman, Owen L; Pepe, Salvatore; Cao, Anh H; Qin, Chengxue; Ritchie, Rebecca H

    2007-01-01

    The objective was a comprehensive investigation of the mechanisms and sites of resveratrol cardioprotection during and following ischemia-reperfusion (I-R) injury, and to determine whether direct preservation of cardiomyocytes is an important site of cardioprotection. We now provide the first definitive evidence that resveratrol specifically protects cardiomyocytes from I-R injury via a combination of suppression of superoxide levels and activation of potassium channels. This protection is apparent whether resveratrol is present for the full duration of the insult or only on recovery. In addition, resveratrol improved postischemic recovery of left ventricular contractile function, attenuated myocardial injury, and increased myocardial activation of the survival kinase Akt in the intact heart. Furthermore, resveratrol elicited direct concentration-dependent protective actions on the vasculature (vasorelaxation, superoxide suppression) and enhanced endothelium-dependent vasodilatation. Resveratrol thus targets a number of consequences of myocardial I-R, including release of reactive oxygen species, loss of recovery of contractile function, and impaired endothelium-dependent vasodilatation. Previous evidence indicates that resveratrol elicits potent preconditioning in the heart. Given that myocardial ischemic events are often unpredictable in humans, the findings that resveratrol protection is also evident when administered during and/or after the insult adds new dimensions to the clinical potential of resveratrol.

  14. Investigation of confined placental mosaicism (CPM) at multiple sites in post-delivery placentas derived through intracytoplasmic sperm injection (ICSI).

    PubMed

    Minor, Agata; Harmer, Karynn; Peters, Nicole; Yuen, Basil Ho; Ma, Sai

    2006-01-01

    Although earlier studies on pregnancies derived through intracytoplasmic sperm injection (ICSI) reported increased non-mosaic aneuploidy among ICSI children, undetected mosaicism, such as confined placental mosaicism (CPM) has not been evaluated. We investigated the incidence of CPM in post-delivery placentas derived from ICSI, evaluated whether CPM was increased and whether it was a contributing factor to negative pregnancy outcome. [Fifty-one post-delivery placentas were collected from patients who underwent ICSI with a normal or negative pregnancy outcome]. Trophoblast and chorionic stroma from three sites were analyzed by comparative genomic hybridization (CGH) and flow cytometry. Detected abnormalities were confirmed by fluorescence in situ hybridization (FISH). The incidence of CPM in the ICSI population was compared to the general population from published data. We detected three cases of CPM in our study. One abnormality was found by CGH analysis; partial trisomy 7q and a partial monosomy Xp limited to the trophoblast at two sites. The abnormality was associated with a child affected by spina bifida. Two cases of mosaic tetraploidy were observed by flow cytometry in pregnancies with a normal outcome. All three abnormalities were confirmed by FISH analysis. The incidence of CPM in the ICSI study population was 5.88% (3/51), which was not statistically different from published reports in the general population (5.88% (42/714), Chi square, P > 0.05). The post-ICSI population was not at risk for CPM in this study.

  15. A study of the undisturbed mid-latitude ionosphere using simultaneous multiple site ionosonde measurements during the Sundial-86 campaign

    SciTech Connect

    Sica, R.J. ); Schunk, R.W. ); Willkinson, P.J. )

    1990-06-01

    The Sundial-86 campaign obtained simultaneous ionosonde measurements of N{sub m}F2 from 41 mid-latitude sites geographically dispersed around the world from September 21 to October 5, 1986. A three-dimensional, time-dependent model of Earth's ionoshpere has been used to fit the variations in N{sub m}F2 obtained from these sites. The model successfully reproduced the measurements in both the northern and southern hemispheres using the vertical plasma drift as a free parameter. During geomagnetically quiet conditions this drift is primarily due to the meridional neutral wind. The vertical plasma drift required to fit the data is consistent from location to location and the deduced meridional neutral wind is also consistent with previous neutral wind measurements and models. However, it is shown that a lack of knowledge of the variation of the O{sup +} flux at the top of the atmosphere introduces a large uncertainty in the deduced thermospheric winds.

  16. HPC Analysis of Multiple Binding Sites Communication and Allosteric Modulations in Drug Design: The HSP Case Study.

    PubMed

    Chiappori, Federica; Milanesi, Luciano; Merelli, Ivan

    2016-01-01

    Allostery is a long-range macromolecular mechanism of internal regulation, in which the binding of a ligand in an allosteric site induces distant conformational changes in a distant portion of the protein, modifying its activity. From the drug design point of view, this mechanism can be exploited to achieve important therapeutic effects, since ligands able to bind allosteric sites may be designed to regulate target proteins. Computational tools are a valid support in this sense, since they allow the characterization of allosteric communications within proteins, which are essential to design modulator ligands. While considering long-range interactions in macromolecules, the principal drug design tool available to researcher is molecular dynamics, and related applications, since it allows the evaluation of conformational changes of a protein bound to a ligand. In particular, all-atoms molecular dynamics is suitable to verify the internal mechanisms that orchestrate allosteric communications, in order to identify key residues and internal pathways that modify the protein behaviour. The problem is that these techniques are heavily time-consuming and computationally intensive, thus high performance computing systems, including parallel computing and GPU-accelerated computations, are necessary to achieve results in a reasonable time. In this review, we will discuss how it is possible to exploit in silico approaches to characterize allosteric modulations and long-range interactions within proteins, describing the case study of the Heat Shock Proteins, a class of chaperons regulated by stress conditions, which is particularly important since it is involved in many cancers and neurodegenerative diseases.

  17. Non-homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process.

    PubMed

    Rulten, Stuart L; Grundy, Gabrielle J

    2017-03-01

    Non-homologous end-joining (NHEJ) is the dominant means of repairing chromosomal DNA double strand breaks (DSBs), and is essential in human cells. Fifteen or more proteins can be involved in the detection, signalling, synapsis, end-processing and ligation events required to repair a DSB, and must be assembled in the confined space around the DNA ends. We review here a number of interaction points between the core NHEJ components (Ku70, Ku80, DNA-PKcs, XRCC4 and Ligase IV) and accessory factors such as kinases, phosphatases, polymerases and structural proteins. Conserved protein-protein interaction sites such as Ku-binding motifs (KBMs), XLF-like motifs (XLMs), FHA and BRCT domains illustrate that different proteins compete for the same binding sites on the core machinery, and must be spatially and temporally regulated. We discuss how post-translational modifications such as phosphorylation, ADP-ribosylation and ubiquitinylation may regulate sequential steps in the NHEJ pathway or control repair at different types of DNA breaks.

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

    PubMed Central

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

    2016-01-01

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

  19. Implant site preparation using a single bur versus multiple drilling steps: 4-month post-loading results of a multicenter randomised controlled trial.

    PubMed

    Guazzi, Paolo; Grandi, Tommaso; Grandi, Giovanni

    2015-01-01

    To compare the clinical outcome of implants inserted in sites prepared with a simplified protocol consisting of one single drill versus multiple conventional drilling steps. In two private clinics, 40 patients, requiring one single implant and having a residual bone height of at least 10 mm and a thickness of at least 5 mm measured on computerised tomography (CT) scans, were randomised after flap elevation to have the implant site prepared using a single drilling step with a newly designed tapered-cylinder drill (1-drill group) or a conventional procedure with multiple drills (multiple-drill group). Implants were left to heal non-submerged for 3 months and then they were loaded with a final metal-ceramic crown. Outcome measures were: implant failure; any complications; peri-implant marginal bone level changes assessed by a blinded outcome assessor; operation time; operator preference and post-surgical pain, swelling and analgesic consumption. All patients were followed up to 4 months after implant loading. Twenty patients were randomised to the 1-drill group and 20 patients to the multiple-drill group. No implant failed and no complications occurred. Four months after loading, implants in the 1-drill group lost 0.54 mm of peri-implant bone versus 0.41 mm for the implants in the multiple-drill group. There were no statistically significant differences for marginal bone level changes between the two groups (difference 0.13 mm, 95% CI -0.21; 0.47, P = 0.108). Less time which was statistically significant (3.66 mins, 95% CI 2.69; 4.63, P < 0.0001) was required to place the implant with the single bur. Both operators always preferred the single bur technique. Postoperatively, patients in the 1-drill group vs patients in the multiple-drill group reported statistically significant differences for pain level (difference 27.5, 95% CI 3.3; 51.7, P < 0.0001), number of days in which the swelling persisted (difference 3.4, 95% CI 2.4; 4.4, P < 0.0001) and the number of

  20. A multiple-method approach to flood assessment at a low-level radioactive waste site in southern Nevada

    SciTech Connect

    Miller, J.J.; Gustafson, D.L.; Schmeltzer, J.S.

    1994-12-31

    Flood hazard analysis on alluvial fans using Federal Emergency Management Agency (FEMA) method are not limited to the FEMA Alluvial Fan Methodology (FEMA AFM). Flood hazard delineations using a combination of methods provide a more thorough assessment that using only the FEMA AFM. Other FEMA-accepted methods, such as the HEC-2 model for shallow concentrated flow and the Manning Equation for sheetflow, may be more appropriate. A flood assessment using a multiple-method approach was performed to determine the 100-year flood hazard in this arid region. Understanding the limitations and assumptions of these methods is important to determine which method is applicable and when a method can provide reasonable results.

  1. Quantitative detection of multiple fluorophore sites as a tool for diagnosis and monitoring disease progression in salivary glands

    NASA Astrophysics Data System (ADS)

    Gannot, Israel; Bonner, Robert F.; Gannot, Gallya; Fox, Philip C.; You, Joon S.; Waynant, Ronald W.; Gandjbakhche, Amir H.

    1997-08-01

    A series of fluorescent surface images were obtained from physical models of localized fluorophores embedded at various depths and separations in tissue phantoms. Our random walk theory was applied to create an analytical model of multiple flurophores embedded in tissue-like phantom. Using this model, from acquired set of surface images, the location of the fluorophores was reconstructed and compared it to their known 3-D distributions. A good correlation was found, and the ability to resolve fluorophores as a function of depth and separation was determined. In parallel in in-vitro study, specific coloring of sections of minor salivary glands was also demonstrated. These results demonstrate the possibility of using inverse methods to reconstruct unknown locations and concentrations of optical probes specifically bound to infiltrating lymphocytes in minor salivary glands of patients with Sjogren's syndrome.

  2. Previous Fractures at Multiple Sites Increase the Risk for Subsequent Fractures: The Global Longitudinal Study of Osteoporosis in Women

    PubMed Central

    Gehlbach, Stephen; Saag, Kenneth G.; Adachi, Jonathan D.; Hooven, Fred H.; Flahive, Julie; Boonen, Steven; Chapurlat, Roland D.; Compston, Juliet E.; Cooper, Cyrus; Díez-Perez, Adolfo; Greenspan, Susan L.; LaCroix, Andrea Z.; Netelenbos, J. Coen; Pfeilschifter, Johannes; Rossini, Maurizio; Roux, Christian; Sambrook, Philip N.; Silverman, Stuart; Siris, Ethel S.; Watts, Nelson B.; Lindsay, Robert

    2016-01-01

    Previous fractures of the hip, spine, or wrist are well-recognized predictors of future fracture, but the role of other fracture sites is less clear. We sought to assess the relationship between prior fracture at 10 skeletal locations and incident fracture. The Global Longitudinal Study of Osteoporosis in Women (GLOW) is an observational cohort study being conducted in 17 physician practices in 10 countries. Women ≥ 55 years answered questionnaires at baseline and at 1 and/or 2 years (fractures in previous year). Of 60,393 women enrolled, follow-up data were available for 51,762. Of these, 17.6%, 4.0%, and 1.6% had suffered 1, 2, or ≥3 fractures since age 45. During the first 2 years of follow-up, 3149 women suffered 3683 incident fractures. Compared with women with no prior fractures, women with 1, 2, or ≥ 3 prior fractures were 1.8-, 3.0-, and 4.8-fold more likely to have any incident fracture; those with ≥3 prior fractures were 9.1-fold more likely to sustain a new vertebral fracture. Nine of 10 prior fracture locations were associated with an incident fracture. The strongest predictors of incident spine and hip fractures were prior spine fracture (hazard ratio 7.3) and hip (hazard ratio 3.5). Prior rib fractures were associated with a 2.3-fold risk of subsequent vertebral fracture, previous upper leg fracture predicted a 2.2-fold increased risk of hip fracture; women with a history of ankle fracture were at 1.8-fold risk of future fracture of a weight-bearing bone. Our findings suggest that a broad range of prior fracture sites are associated with an increased risk of incident fractures, with important implications for clinical assessments and risk model development. PMID:22113888

  3. Microbiota at Multiple Body Sites during Pregnancy in a Rural Tanzanian Population and Effects of Moringa-Supplemented Probiotic Yogurt.

    PubMed

    Bisanz, Jordan E; Enos, Megan K; PrayGod, George; Seney, Shannon; Macklaim, Jean M; Chilton, Stephanie; Willner, Dana; Knight, Rob; Fusch, Christoph; Fusch, Gerhard; Gloor, Gregory B; Burton, Jeremy P; Reid, Gregor

    2015-08-01

    The nutritional status of pregnant women is vital for healthy outcomes and is a concern for a large proportion of the world's population. The role of the microbiota in pregnancy and nutrition is a promising new area of study with potential health ramifications. In many African countries, maternal and infant death and morbidity are associated with malnutrition. Here, we assess the influence of probiotic yogurt containing Lactobacillus rhamnosus GR-1, supplemented with Moringa plant as a source of micronutrients, on the health and oral, gut, vaginal, and milk microbiotas of 56 pregnant women in Tanzania. In an open-label study design, 26 subjects received yogurt daily, and 30 were untreated during the last two trimesters and for 1 month after birth. Samples were analyzed using 16S rRNA gene sequencing, and dietary recalls were recorded. Women initially categorized as nourished or undernourished consumed similar calories and macronutrients, which may explain why there was no difference in the microbiota at any body site. Consumption of yogurt increased the relative abundance of Bifidobacterium and decreased Enterobacteriaceae in the newborn feces but had no effect on the mother's microbiota at any body site. The microbiota of the oral cavity and GI tract remained stable over pregnancy, but the vaginal microbiota showed a significant increase in diversity leading up to and after birth. In summary, daily micronutrient-supplemented probiotic yogurt provides a safe, affordable food for pregnant women in rural Tanzania, and the resultant improvement in the gut microbial profile of infants is worthy of further study.

  4. Microbiota at Multiple Body Sites during Pregnancy in a Rural Tanzanian Population and Effects of Moringa-Supplemented Probiotic Yogurt

    PubMed Central

    Bisanz, Jordan E.; Enos, Megan K.; PrayGod, George; Seney, Shannon; Macklaim, Jean M.; Chilton, Stephanie; Willner, Dana; Knight, Rob; Fusch, Christoph; Fusch, Gerhard; Gloor, Gregory B.; Burton, Jeremy P.

    2015-01-01

    The nutritional status of pregnant women is vital for healthy outcomes and is a concern for a large proportion of the world's population. The role of the microbiota in pregnancy and nutrition is a promising new area of study with potential health ramifications. In many African countries, maternal and infant death and morbidity are associated with malnutrition. Here, we assess the influence of probiotic yogurt containing Lactobacillus rhamnosus GR-1, supplemented with Moringa plant as a source of micronutrients, on the health and oral, gut, vaginal, and milk microbiotas of 56 pregnant women in Tanzania. In an open-label study design, 26 subjects received yogurt daily, and 30 were untreated during the last two trimesters and for 1 month after birth. Samples were analyzed using 16S rRNA gene sequencing, and dietary recalls were recorded. Women initially categorized as nourished or undernourished consumed similar calories and macronutrients, which may explain why there was no difference in the microbiota at any body site. Consumption of yogurt increased the relative abundance of Bifidobacterium and decreased Enterobacteriaceae in the newborn feces but had no effect on the mother's microbiota at any body site. The microbiota of the oral cavity and GI tract remained stable over pregnancy, but the vaginal microbiota showed a significant increase in diversity leading up to and after birth. In summary, daily micronutrient-supplemented probiotic yogurt provides a safe, affordable food for pregnant women in rural Tanzania, and the resultant improvement in the gut microbial profile of infants is worthy of further study. PMID:25979893

  5. The Arabidopsis PR-1 Promoter Contains Multiple Integration Sites for the Coactivator NPR1 and the Repressor SNI1[W

    PubMed Central

    Pape, Sebastian; Thurow, Corinna; Gatz, Christiane

    2010-01-01

    Systemic acquired resistance is a broad-spectrum plant immune response involving massive transcriptional reprogramming. The Arabidopsis (Arabidopsis thaliana) PATHOGENESIS-RELATED-1 (PR-1) gene has been used in numerous studies to elucidate transcriptional control mechanisms regulating systemic acquired resistance. WRKY transcription factors and basic leucine zipper proteins of the TGA family regulate the PR-1 promoter by binding to specific cis-elements. In addition, the promoter is under the control of two proteins that do not directly contact the DNA: the positive regulator NONEXPRESSOR OF PR GENES1 (NPR1), which physically interacts with TGA factors, and the repressor SUPPRESSOR OF NPR1, INDUCIBLE1 (SNI1). In this study, we analyzed the importance of the TGA-binding sites LS5 and LS7 and the WKRY box LS4 for regulation by NPR1 and SNI1. In the absence of LS5 and LS7, NPR1 activates the PR-1 promoter through a mechanism that requires LS4. Since transcriptional activation of WRKY genes is under the control of NPR1 and since LS4 is not sufficient for the activation of a truncated PR-1 promoter by the effector protein NPR1-VP16 in transient assays, it is concluded that the LS4-dependent activation of the PR-1 promoter is indirect. In the case of NPR1 acting directly through TGA factors at its target promoters, two TGA-binding sites are necessary but not sufficient for NPR1 function in transgenic plants and in the NPR-VP16-based trans-activation assay in protoplasts. SNI1 exerts its negative effect in the noninduced state by targeting unknown proteins associated with sequences between bp −816 and −573. Under induced conditions, SNI1 negatively regulates the function of WRKY transcription factors binding to WKRY boxes between bp −550 and −510. PMID:20935179

  6. Two versatile shuttle vectors for Thermus thermophilus-Escherichia coli containing multiple cloning sites, lacZα gene and kanamycin or hygromycin resistance marker.

    PubMed

    Fujita, Atsushi; Misumi, Yoshio; Koyama, Yoshinori

    2012-05-01

    Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZα gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus.

  7. Utility of multiple chemical techniques in archaeological residential mobility studies: case studies from Tiwanaku- and Chiribaya-affiliated sites in the Andes.

    PubMed

    Knudson, Kelly J; Price, T Douglas

    2007-01-01

    In the south central Andes, archaeologists have long debated the extent of Tiwanaku colonization during the Middle Horizon (AD 500-1000). We tested the hypotheses regarding the nature of Tiwanaku influence using strontium isotope, trace element concentration, and oxygen isotope data from archaeological human tooth enamel and bone from Tiwanaku- and Chiribaya-affiliated sites in the south central Andes. Strontium isotope analysis of 25 individuals buried at the Tiwanaku-affiliated Moquegua Valley site of Chen Chen demonstrates that it was likely a Tiwanaku colony. In contrast, no immigrants from the Lake Titicaca Basin were present in 27 individuals analyzed from the San Pedro de Atacama cemeteries of Coyo Oriental, Coyo-3, and Solcor-3; it is likely that these sites represent economic and religious alliances, but not colonies. However, strontium isotope analysis alone cannot distinguish movement between the Tiwanaku- and Chiribaya-affiliated sites in the Moquegua and Ilo Valleys of southern Peru. Analyzing oxygen isotope and trace element concentration data and comparing it with strontium isotope data from the same individuals provides a more detailed picture of residential mobility in the Tiwanaku and Chiribaya polities. In addition to monitoring diagenetic contamination, trace element concentration data identified movement during adulthood for certain individuals. However, these data could not distinguish movement between the Moquegua and Ilo Valleys. While oxygen isotope data could clearly distinguish the high-altitude sites from others, more data is needed to characterize the local oxygen isotope ratios of these regions. These data demonstrate the potential for archaeological reconstruction of residential mobility through multiple lines of evidence. (c) 2006 Wiley-Liss, Inc

  8. Daidzein together with high calcium preserve bone mass and biomechanical strength at multiple sites in ovariectomized mice.

    PubMed

    Fonseca, Debbie; Ward, Wendy E

    2004-08-01

    As the prevalence of osteoporosis is increasing, and the adverse effects of hormone replacement therapy are evident, women are searching for natural alternatives such as soy isoflavones to help prevent postmenopausal osteoporosis. Daidzein is one of the most abundant isoflavones present in soy and it is unique as it can be further metabolized to equol, a compound with greater estrogenic activity than other isoflavones. The objective of this study was to determine the effects of purified daidzein in combination with high calcium (Ca) on preserving femur and lumbar vertebrae (LV1-LV4) bone mineral density (BMD) and biomechanical bone strength at three different sites (femur midpoint, femur neck and LV3) in ovariectomized mice. Sham (SH) mice (n = 12) received control diet (AIN93G) containing 2 g Ca/kg diet and ovariectomized mice were randomized to 1 of 6 groups (n = 12/group): OVX (2 g Ca/kg diet), HCa (25 g Ca/kg diet), HD (2 g Ca + 200 mg daidzein/kg diet), HDCa (25 g Ca + 200 mg daidzein/kg diet), LD (2 g Ca + 100 mg daidzein/kg diet) or LDCa (25 g Ca + 100 mg daidzein/kg diet) for 12 weeks. HDCa preserved femur and vertebrae BMD and biomechanical bone strength (at all three sites) compared to the OVX group, however, only femur yield load (at midpoint) was preserved to a level that was greater (P < 0.05) than HCa alone. Mice fed HD diet had greater (P < 0.05) femur BMD than OVX group, however, daidzein alone (HD) did not appear to preserve trabecular bone (i.e., vertebrae BMD and vertebra peak load). All mice fed daidzein produced equol and there were no uterotrophic effects of daidzein at either dose. Both daidzein and Ca attenuated the increase in serum IL-1beta observed in the OVX group. The results from this study suggest that the combination of daidzein and high Ca favorably affect cortical and trabecular bone as indicated by femur and lumbar vertebrae BMD and biomechanical strength but much of this effect is mediated by the high Ca diet. Further

  9. Geochemistry of ferromanganese nodules from DOMES site a, Northern Equatorial Pacific: Multiple diagenetic metal sources in the deep sea

    USGS Publications Warehouse

    Calvert, S.E.; Piper, D.Z.

    1984-01-01

    The major and minor element composition of ferromanganese nodules from DOMES Site A has been determined by X-ray fluorescence methods. Three phases appear to control the bulk compositions: Mn and Fe oxyhydroxides and aluminosilicates. Relatively wide compositional variations are evident throughout the area. Nodules with high Mn/Fe ratios, high Cu, Mg, Mo, Ni and Zn concentrations and high todorokite/??-MnO2 ratios have granular surface textures and are confined to an east-west trending depression with thin Quaternary sediment cover. Nodules with low Mn/Fe ratios, high concentrations of As, Ca, Ce, Co, La, P, Sr, Ti, V, Y and Zr and low todorokite/??-MnO2 ratios have smooth surfaces and are confined to shallower areas with relatively thick Quaternary sediment to the north and south of the depression. All nodules in the area have compositions which are influenced by diagenesis, but those with the most marked diagenetic signature (high Mn/Fe and Cu/Ni ratios, low Ce/La ratios and more todorokite) are found in areas of very slow or non-existent sedimentation; many of these nodules are actually in contact with outcropping Tertiary sediment. This paradox may be resolved by postulating, by analogy with some shallow-water occurrences, that the nodules accrete from bottom waters which have enhanced particulate and dissolved metal contents derived from diagenetic reaction in areas remote from the site of nodule formation. The metals are supplied in a bottom flow (probably Antarctic Bottom Water) which also erodes, or prevents modern sedimentation in, the depression. Nodules on the flanks of the depression are not evidently affected by this flow and derive at least pan of their constituent metals from diagenetic reaction in the underlying Quaternary sediment. Apparently, abyssal diagenetic nodules can have an immediate and a remote diagenetic metal source. Metal fluxes derived from pore water dissolved metal gradients may not be relevant to particular accreting nodules if a

  10. Significant geographic gradients in particulate sulfate over Japan determined from multiple-site measurements and a chemical transport model: Impacts of transboundary pollution from the Asian continent

    NASA Astrophysics Data System (ADS)

    Aikawa, Masahide; Ohara, Toshimasa; Hiraki, Takatoshi; Oishi, Okihiro; Tsuji, Akihiro; Yamagami, Makiko; Murano, Kentaro; Mukai, Hitoshi

    2010-01-01

    We found a significant geographic gradient (longitudinal and latitudinal) in the sulfate (SO 42-) concentrations measured at multiple sites over the East Asian Pacific Rim region. Furthermore, the observed gradient was well reproduced by a regional chemical transport model. The observed and modeled SO 42- concentrations were higher at the sites closer to the Asian continent. The concentrations of SO 42- from China as calculated by the model also showed the fundamental features of the longitudinal/latitudinal gradient. The proportional contribution of Chinese SO 42- to the total in Japan throughout the year was above 50-70% in the control case, using data for Chinese sulfur dioxide (SO 2) emission from the Regional Emission Inventory in Asia (40-60% in the low Chinese emissions case, using Chinese SO 2 emissions data from the State Environmental Protection Administration of China), with a winter maximum of approximately 65-80%, although the actual concentrations of SO 42- from China were highest in summer. The multiple-site measurements and the model analysis strongly suggest that the SO 42- concentrations in Japan were influenced by the outflow from the Asian continent, and this influence was greatest in the areas closer to the Asian continent. In contrast, we found no longitudinal/latitudinal gradient in SO 2 concentrations; instead SO 2 concentrations were significantly correlated with local SO 2 emissions. Our results show that large amounts of particulate sulfate are transported over long distances from the East Asian Pacific Rim region, and consequently the SO 42- concentrations in Japan are controlled by the transboundary outflow from the Asian continent.

  11. First imaging and identification of a noctilucent cloud from multiple sites in Hokkaido (43.2-44.4°N), Japan

    NASA Astrophysics Data System (ADS)

    Suzuki, Hidehiko; Sakanoi, Kazuyo; Nishitani, Nozomu; Ogawa, Tadahiko; Ejiri, Mitsumu K.; Kubota, Minoru; Kinoshita, Takenori; Murayama, Yasuhiro; Fujiyoshi, Yasushi

    2016-11-01

    Simultaneous imaging observations of a noctilucent cloud (NLC) from five sites in Hokkaido, Japan (43.17-45.36°N), were successfully carried out using digital cameras in the early hours of the morning (around 02:00 LST) on June 21, 2015. This is the first NLC event that has been captured from multiple sites in Japan. The simultaneous images obtained from multiple sites made it possible to calculate the exact altitude (=83.9 ± 0.1 km) and spatial distribution (47.5-50.0°N and 143.0-147.5°E) of the NLC by triangulation and image correlation methods. Based on a comparison of atmospheric parameters of the upper mesosphere provided by satellites and a middle-frequency (MF) radar in northern Hokkaido (Wakkanai) with the cloud distribution obtained from the Aeronomy of Ice in the Mesosphere satellite, this particular event is considered to be the result of southward advection of the NLC from a higher-latitude (i.e., colder) region. Anomalies in the upper mesospheric temperature of the northern hemispheric summer in 2015 were examined using AURA satellite data, because this is the first NLC event that has been identified in Japan. However, no remarkable temperature variations relative to other years were found in upper mesosphere. Based on a comparison between the NLC period and the record of sky conditions archived by the Japan Meteorological Agency, a high percentage of cloud (especially low-level) cover during the summer in Hokkaido cannot be ruled out as a possible reason why the NLC had not previously been sighted in Hokkaido.[Figure not available: see fulltext.

  12. Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

    PubMed

    Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

    2016-03-01

    Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

  13. FAst MEtabolizer (FAME): A rapid and accurate predictor of sites of metabolism in multiple species by endogenous enzymes.

    PubMed

    Kirchmair, Johannes; Williamson, Mark J; Afzal, Avid M; Tyzack, Jonathan D; Choy, Alison P K; Howlett, Andrew; Rydberg, Patrik; Glen, Robert C

    2013-11-25

    FAst MEtabolizer (FAME) is a fast and accurate predictor of sites of metabolism (SoMs). It is based on a collection of random forest models trained on diverse chemical data sets of more than 20 000 molecules annotated with their experimentally determined SoMs. Using a comprehensive set of available data, FAME aims to assess metabolic processes from a holistic point of view. It is not limited to a specific enzyme family or species. Besides a global model, dedicated models are available for human, rat, and dog metabolism; specific prediction of phase I and II metabolism is also supported. FAME is able to identify at least one known SoM among the top-1, top-2, and top-3 highest ranked atom positions in up to 71%, 81%, and 87% of all cases tested, respectively. These prediction rates are comparable to or better than SoM predictors focused on specific enzyme families (such as cytochrome P450s), despite the fact that FAME uses only seven chemical descriptors. FAME covers a very broad chemical space, which together with its inter- and extrapolation power makes it applicable to a wide range of chemicals. Predictions take less than 2.5 s per molecule in batch mode on an Ultrabook. Results are visualized using Jmol, with the most likely SoMs highlighted.

  14. Hsp90·Cdc37 Complexes with Protein Kinases Form Cooperatively with Multiple Distinct Interaction Sites.

    PubMed

    Eckl, Julia M; Scherr, Matthias J; Freiburger, Lee; Daake, Marina A; Sattler, Michael; Richter, Klaus

    2015-12-25

    Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

    PubMed Central

    Hou, Yong; Li, Jianwei; Li, Yi; Dong, Zhaoming; Xia, Qingyou; Yuan, Y Adam

    2014-01-01

    In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level. PMID:24639361

  16. Comparative Genomic Hybridization of Human Malignant Gliomas Reveals Multiple Amplification Sites and Nonrandom Chromosomal Gains and Losses

    PubMed Central

    Schròck, Evelin; Thiel, Gundula; Lozanova, Tanka; du Manoir, Stanislas; Meffert, Marie-Christine; Jauch, Anna; Speicher, Michael R.; Nürnberg, Peter; Vogel, Siegfried; Janisch, Werner; Donis-Keller, Helen; Ried, Thomas; Witkowski, Regine; Cremer, Thomas

    1994-01-01

    Nine human malignant gliomas (2 astrocytomas grade III and 7 glioblastomas) were analyzed using comparative genomic hybridization (CGH). In addition to the amplification of the EGFR gene at 7p12 in 4 of 9 cases, six new amplification sites were mapped to 1q32, 4q12, 7q21.1, 7q21.2-3, 12p, and 22q12. Nonrandom chromosomal gains and losses were identified with overrepresentation of chromosome 7 and underrepresentation of chromosome 10 as the most frequent events (1 of 2 astrocytomas, 7 of 7 glioblastomas). Gain of a part or the whole chromosome 19 and losses of chromosome bands 9pter-23 and 22q13 were detected each in five cases. Loss of chromosome band 17p13 and gain of chromosome 20 were revealed each in three cases. The validity of the CGH data was confirmed using interphase cytogenetics with YAC clones, chromosome painting in tumor metaphase spreads, and DNA fingerprinting. A comparison of CGH data with the results of chromosome banding analyses indicates that metaphase spreads accessible in primary tumor cell cultures may not represent the clones predominant in the tumor tissue ImagesFigure 1Figure 4Figure 6 PMID:8203461

  17. Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites.

    PubMed

    Norton, Gareth J; Pinson, Shannon R M; Alexander, Jill; McKay, Susan; Hansen, Helle; Duan, Gui-Lan; Rafiqul Islam, M; Islam, Shofiqul; Stroud, Jacqueline L; Zhao, Fang-Jie; McGrath, Steve P; Zhu, Yong-Guan; Lahner, Brett; Yakubova, Elena; Guerinot, Mary Lou; Tarpley, Lee; Eizenga, Georgia C; Salt, David E; Meharg, Andrew A; Price, Adam H

    2012-02-01

    • Inorganic arsenic (As(i) ) in rice (Oryza sativa) grains is a possible threat to human health, with risk being strongly linked to total dietary rice consumption and consumed rice As(i) content. This study aimed to identify the range and stability of genetic variation in grain arsenic (As) in rice. • Six field trials were conducted (one each in Bangladesh and China, two in Arkansas, USA over 2 yr, and two in Texas, USA comparing flooded and nonflood treatments) on a large number of common rice cultivars (c. 300) representing genetic diversity among international rice cultivars. • Within each field there was a 3-34 fold range in grain As concentration which varied between rice subpopulations. Importantly, As(i) correlated strongly with total As among a subset of 40 cultivars harvested in Bangladesh and China. • Genetic variation at all field sites was a large determining factor for grain As concentration, indicating that cultivars low in grain As could be developed through breeding. The temperate japonicas exhibited lower grain As compared with other subpopulations. Effects for year, location and flooding management were also statistically significant, suggesting that breeding strategies must take into account environmental factors.

  18. Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

    PubMed Central

    Millard, J T; Weidner, M F; Kirchner, J J; Ribeiro, S; Hopkins, P B

    1991-01-01

    A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency. Images PMID:1903204

  19. Multiple phosphate positions in the catalytic site of glycogen phosphorylase: structure of the pyridoxal-5'-pyrophosphate coenzyme-substrate analog.

    PubMed Central

    Sprang, S. R.; Madsen, N. B.; Withers, S. G.

    1992-01-01

    The three-dimensional structure of an R-state conformer of glycogen phosphorylase containing the coenzyme-substrate analog pyridoxal-5'-diphosphate at the catalytic site (PLPP-GPb) has been refined by X-ray crystallography to a resolution of 2.87 A. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R-state PLPP-GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609-616), the tertiary structures differ in that the two domains of the PLPP-GPb subunits are rotated apart by 5 degrees relative to the T-state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R-state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R-state. PMID:1304389

  20. Multiple phosphorylation sites of DNA polymerase alpha-primase cooperate to regulate the initiation of DNA replication in vitro.

    PubMed

    Schub, O; Rohaly, G; Smith, R W; Schneider, A; Dehde, S; Dornreiter, I; Nasheuer, H P

    2001-10-12

    DNA polymerase alpha-primase (pol-prim) is the only enzyme that can start DNA replication de novo. The 180-kDa (p180) and 68-kDa (p68) subunits of the human four-subunit enzyme are phosphorylated by Cyclin-dependent kinases (Cdks) in a cell cycle-dependent manner. Cyclin A-Cdk2 physically interacts with pol-prim and phosphorylates N-terminal amino acids of the p180 and the p68 subunits, leading to an inhibition of pol-prim in initiating cell-free SV40 DNA replication. Mutation of conserved putative Cdk phosphorylation sites in the N terminus of human p180 and p68 reduced their phosphorylation by Cyclin A-Cdk2 in vitro. In contrast to wild-type pol-prim these mutants were no longer inhibited by Cyclin A-Cdk2 in the initiation of viral DNA replication. Importantly, rather than inhibiting it, Cyclin A-Cdk2 stimulated the initiation activity of pol-prim containing a triple N-terminal alanine mutant of the p180 subunit. Together these results suggest that Cyclin A-Cdk2 executes both stimulatory and inhibitory effects on the activity of pol-prim in initiating DNA replication.

  1. Wild leafy vegetable use and knowledge across multiple sites in Morocco: a case study for transmission of local knowledge?

    PubMed Central

    2014-01-01

    Background There are few publications on the use and diversity of wild leafy vegetables (WLVs) in Morocco. In order to address this gap, we conducted ethnobotanical field work in Taounate, Azilal and El House regions. Methods Ethnobotanical collections, free listing, qualitative interviews and a 7 day food frequency questionnaire. Results More than 30 species in 23 genera of WLV were identified. Of these 4 had not previously recorded as WLVs used in Morocco in the literature. WLVs were used by 84% of households surveyed in Taounate (N = 61, in March 2005), and were used up to 4 times a week. Qualitative data revealed both positive and negative perceptions of WLVs and detailed knowledge about preparation among women. The greatest diversity of WLV knowledge and use was in the Rif Mountains (Taounate). There was significant variation in nomenclature and salience of WLVs, not only between regions, but also between villages in the same region. Within the same region (or even village) different local names were used for a given species or genus, and different species were identified by the same local name (including species from different botanical families). Data showed greater overlap in knowledge among villages using the same market. Conclusion We believe the results suggest that markets are important sites for WLV knowledge transmission. PMID:24708730

  2. Effect of application sites and multiple doses on nicotine pharmacokinetics in healthy male Japanese smokers following application of the transdermal nicotine patch.

    PubMed

    Sobue, Satoshi; Sekiguchi, Kaneo; Kikkawa, Hironori; Irie, Shin

    2005-12-01

    The transdermal nicotine patch, which contains 25 mg nicotine per 30 cm(2), is designed to deliver approximately 15 mg nicotine to the blood circulation in 16 hours of application for the treatment of smoking cessation. It was applied to 3 different skin sites (upper arm, abdomen, and back) to examine regional variations in percutaneous nicotine absorption in a single-dose, 3-period, crossover study involving 9 healthy male Japanese smokers. Nicotine pharmacokinetics during once-daily application of the transdermal nicotine patch for 5 days was also investigated in 10 healthy smokers. There were statistically significant effects of application sites on percutaneous nicotine absorption. The ratios (90% confidence intervals) of AUC and C(max) for comparison to the upper arm were 102% (88, 117%) and 106% (95, 119%) for the back and 75% (65, 87%) and 75% (66, 84%) for the abdomen, respectively. These suggest that systemic exposure after application to the upper arm was greater compared with the abdomen but equivalent to the back. Following multiple doses, linear pharmacokinetics and no significant accumulation of nicotine concentrations were observed, and steady state was reached by day 2. Only mild itching and erythema were observed at the application sites. The transdermal nicotine patch was well tolerated in both studies.

  3. Multiple-scattering XAS calculations for the characterization of the binuclear (type 3) copper sites of hemocyanins and related model compounds.

    PubMed

    Borghi, Elena; Solari, Pier Lorenzo

    2005-01-01

    Our previous studies on the met- and met-azido-Hc forms from Octopus vulgaris (mollusc) and Carcinus aestuarii (arthropod) at pH 7.5 and on some related binuclear models without X-ray diffraction have considered and resolved some fundamental aspects of their binuclear type-3 copper site (i.e. correct values of the Cu-Cu distances; apical distortion, when present, at the copper site; presence and type of bridging groups). In this contribution the multiple-scattering (MS) calculations performed in order to refine the EXAFS modulation of the absorption spectra are presented. It is only with a composite and advanced approach that some severe problems, mainly deriving from the presence of two absorbing atoms and from the fact that the metal-metal contribution in the absorption spectra overlaps with the Cu-His signals, have been overcome. Results are also presented which indicate the role of the MS calculations in the XANES edge region, and which show how it is possible to extract quantitative information from this zone of the spectrum in order to refine the structure of the site also in the case of a binuclear centre.

  4. The role of multiple hydrogen-bonding groups in specific alcohol binding sites in proteins: insights from structural studies of LUSH.

    PubMed

    Thode, Anna B; Kruse, Schoen W; Nix, Jay C; Jones, David N M

    2008-03-07

    It is now generally accepted that many of the physiological effects of alcohol consumption are a direct result of binding to specific sites in neuronal proteins such as ion channels or other components of neuronal signaling cascades. Binding to these targets generally occurs in water-filled pockets and leads to alterations in protein structure and dynamics. However, the precise interactions required to confer alcohol sensitivity to a particular protein remain undefined. Using information from the previously solved crystal structures of the Drosophila melanogaster protein LUSH in complexes with short-chain alcohols, we have designed and tested the effects of specific amino acid substitutions on alcohol binding. The effects of these substitutions, specifically S52A, T57S, and T57A, were examined using a combination of molecular dynamics, X-ray crystallography, fluorescence spectroscopy, and thermal unfolding. These studies reveal that the binding of ethanol is highly sensitive to small changes in the composition of the alcohol binding site. We find that T57 is the most critical residue for binding alcohols; the T57A substitution completely abolishes binding, while the T57S substitution differentially affects ethanol binding compared to longer-chain alcohols. The additional requirement for a potential hydrogen-bond acceptor at position 52 suggests that both the presence of multiple hydrogen-bonding groups and the identity of the hydrogen-bonding residues are critical for defining an ethanol binding site. These results provide new insights into the detailed chemistry of alcohol's interactions with proteins.

  5. Multiple EBNA1-binding sites within oriPI are required for EBNA1-dependent transactivation of the Epstein-Barr virus C promoter.

    PubMed

    Zetterberg, Henrik; Boreström, Cecilia; Nilsson, Tina; Rymo, Lars

    2004-09-01

    The transactivating function of the oriPI-EBNA1 complex is essential for activation of the Epstein-Barr virus (EBV) C promoter (Cp) in lymphoblastoid cell lines expressing the viral growth programme. Furthermore, the oriPI-EBNA1 complex is believed to play an important role during promoter switching upon primary infection of B-lymphocytes and establishment of latent infection in vivo. Previously, it was shown that six EBNA1-binding sites within oriPI were required for transactivation of the heterologous thymidine kinase promoter. Here, we define the number of EBNA1-binding sites within oriPI necessary for its biological function as EBNA1-dependent Cp enhancer. We show that four EBNA1-binding sites within oriPI lead to significant upregulation of Cp in response to EBNA1 and eight or more to full activation. Thus, multiple EBNA1 homodimers at oriPI are required for the formation of a transcriptionally active Cp complex, a process that involves EBNA1-induced changes in the chromatin structure including DNA looping and nucleosome destabilization.

  6. Floating chip mounting system driven by repulsive force of permanent magnets for multiple on-site SPR immunoassay measurements.

    PubMed

    Horiuchi, Tsutomu; Tobita, Tatsuya; Miura, Toru; Iwasaki, Yuzuru; Seyama, Michiko; Inoue, Suzuyo; Takahashi, Jun-ichi; Haga, Tsuneyuki; Tamechika, Emi

    2012-10-17

    We have developed a measurement chip installation/removal mechanism for a surface plasmon resonance (SPR) immunoassay analysis instrument designed for frequent testing, which requires a rapid and easy technique for changing chips. The key components of the mechanism are refractive index matching gel coated on the rear of the SPR chip and a float that presses the chip down. The refractive index matching gel made it possible to optically couple the chip and the prism of the SPR instrument easily via elastic deformation with no air bubbles. The float has an autonomous attitude control function that keeps the chip parallel in relation to the SPR instrument by employing the repulsive force of permanent magnets between the float and a float guide located in the SPR instrument. This function is realized by balancing the upward elastic force of the gel and the downward force of the float, which experiences a leveling force from the float guide. This system makes it possible to start an SPR measurement immediately after chip installation and to remove the chip immediately after the measurement with a simple and easy method that does not require any fine adjustment. Our sensor chip, which we installed using this mounting system, successfully performed an immunoassay measurement on a model antigen (spiked human-IgG) in a model real sample (non-homogenized milk) that included many kinds of interfering foreign substances without any sample pre-treatment. The ease of the chip installation/removal operation and simple measurement procedure are suitable for frequent on-site agricultural, environmental and medical testing.

  7. Reliability of longitudinal brain volume loss measurements between 2 sites in patients with multiple sclerosis: comparison of 7 quantification techniques.

    PubMed

    Durand-Dubief, F; Belaroussi, B; Armspach, J P; Dufour, M; Roggerone, S; Vukusic, S; Hannoun, S; Sappey-Marinier, D; Confavreux, C; Cotton, F

    2012-11-01

    Brain volume loss is currently a MR imaging marker of neurodegeneration in MS. Available quantification algorithms perform either direct (segmentation-based techniques) or indirect (registration-based techniques) measurements. Because there is no reference standard technique, the assessment of their accuracy and reliability remains a difficult goal. Therefore, the purpose of this work was to assess the robustness of 7 different postprocessing algorithms applied to images acquired from different MR imaging systems. Nine patients with MS were followed longitudinally over 1 year (3 time points) on two 1.5T MR imaging systems. Brain volume change measures were assessed using 7 segmentation algorithms: a segmentation-classification algorithm, FreeSurfer, BBSI, KN-BSI, SIENA, SIENAX, and JI algorithm. Intersite variability showed that segmentation-based techniques and SIENAX provided large and heterogeneous values of brain volume changes. A Bland-Altman analysis showed a mean difference of 1.8%, 0.07%, and 0.79% between the 2 sites, and a wide length agreement interval of 11.66%, 7.92%, and 11.94% for the segmentation-classification algorithm, FreeSurfer, and SIENAX, respectively. In contrast, registration-based algorithms showed better reproducibility, with a low mean difference of 0.45% for BBSI, KN-BSI and JI, and a mean length agreement interval of 1.55%. If SIENA obtained a lower mean difference of 0.12%, its agreement interval of 3.29% was wider. If brain atrophy estimation remains an open issue, future investigations of the accuracy and reliability of the brain volume quantification algorithms are needed to measure the slow and small brain volume changes occurring in MS.

  8. Floating Chip Mounting System Driven by Repulsive Force of Permanent Magnets for Multiple On-Site SPR Immunoassay Measurements

    PubMed Central

    Horiuchi, Tsutomu; Tobita, Tatsuya; Miura, Toru; Iwasaki, Yuzuru; Seyama, Michiko; Inoue, Suzuyo; Takahashi, Jun-ichi; Haga, Tsuneyuki; Tamechika, Emi

    2012-01-01

    We have developed a measurement chip installation/removal mechanism for a surface plasmon resonance (SPR) immunoassay analysis instrument designed for frequent testing, which requires a rapid and easy technique for changing chips. The key components of the mechanism are refractive index matching gel coated on the rear of the SPR chip and a float that presses the chip down. The refractive index matching gel made it possible to optically couple the chip and the prism of the SPR instrument easily via elastic deformation with no air bubbles. The float has an autonomous attitude control function that keeps the chip parallel in relation to the SPR instrument by employing the repulsive force of permanent magnets between the float and a float guide located in the SPR instrument. This function is realized by balancing the upward elastic force of the gel and the downward force of the float, which experiences a leveling force from the float guide. This system makes it possible to start an SPR measurement immediately after chip installation and to remove the chip immediately after the measurement with a simple and easy method that does not require any fine adjustment. Our sensor chip, which we installed using this mounting system, successfully performed an immunoassay measurement on a model antigen (spiked human-IgG) in a model real sample (non-homogenized milk) that included many kinds of interfering foreign substances without any sample pre-treatment. The ease of the chip installation/removal operation and simple measurement procedure are suitable for frequent on-site agricultural, environmental and medical testing. PMID:23202030

  9. The peptide hormone pQDLDHVFLRFamide (crustacean myosuppressin) modulates the Homarus americanus cardiac neuromuscular system at multiple sites.

    PubMed

    Stevens, J S; Cashman, C R; Smith, C M; Beale, K M; Towle, D W; Christie, A E; Dickinson, P S

    2009-12-01

    pQDLDHVFLRFamide is a highly conserved crustacean neuropeptide with a structure that places it within the myosuppressin subfamily of the FMRFamide-like peptides. Despite its apparent ubiquitous conservation in decapod crustaceans, the paracrine and/or endocrine roles played by pQDLDHVFLRFamide remain largely unknown. We have examined the actions of this peptide on the cardiac neuromuscular system of the American lobster Homarus americanus using four preparations: the intact animal, the heart in vitro, the isolated cardiac ganglion (CG), and a stimulated heart muscle preparation. In the intact animal, injection of myosuppressin caused a decrease in heartbeat frequency. Perfusion of the in vitro heart with pQDLDHVFLRFamide elicited a decrease in the frequency and an increase in the amplitude of heart contractions. In the isolated CG, myosuppressin induced a hyperpolarization of the resting membrane potential of cardiac motor neurons and a decrease in the cycle frequency of their bursting. In the stimulated heart muscle preparation, pQDLDHVFLRFamide increased the amplitude of the induced contractions, suggesting that myosuppressin modulates not only the CG, but also peripheral sites. For at least the in vitro heart and the isolated CG, the effects of myosuppressin were dose-dependent (10(-9) to 10(-6) mol l(-1) tested), with threshold concentrations (10(-8)-10(-7) mol l(-1)) consistent with the peptide serving as a circulating hormone. Although cycle frequency, a parameter directly determined by the CG, consistently decreased when pQDLDHVFLRFamide was applied to all preparation types, the magnitudes of this decrease differed, suggesting the possibility that, because myosuppressin modulates the CG and the periphery, it also alters peripheral feedback to the CG.

  10. The peptide hormone pQDLDHVFLRFamide (crustacean myosuppressin) modulates the Homarus americanus cardiac neuromuscular system at multiple sites

    PubMed Central

    Stevens, J. S.; Cashman, C. R.; Smith, C. M.; Beale, K. M.; Towle, D. W.; Christie, A. E.; Dickinson, P. S.

    2009-01-01

    pQDLDHVFLRFamide is a highly conserved crustacean neuropeptide with a structure that places it within the myosuppressin subfamily of the FMRFamide-like peptides. Despite its apparent ubiquitous conservation in decapod crustaceans, the paracrine and/or endocrine roles played by pQDLDHVFLRFamide remain largely unknown. We have examined the actions of this peptide on the cardiac neuromuscular system of the American lobster Homarus americanus using four preparations: the intact animal, the heart in vitro, the isolated cardiac ganglion (CG), and a stimulated heart muscle preparation. In the intact animal, injection of myosuppressin caused a decrease in heartbeat frequency. Perfusion of the in vitro heart with pQDLDHVFLRFamide elicited a decrease in the frequency and an increase in the amplitude of heart contractions. In the isolated CG, myosuppressin induced a hyperpolarization of the resting membrane potential of cardiac motor neurons and a decrease in the cycle frequency of their bursting. In the stimulated heart muscle preparation, pQDLDHVFLRFamide increased the amplitude of the induced contractions, suggesting that myosuppressin modulates not only the CG, but also peripheral sites. For at least the in vitro heart and the isolated CG, the effects of myosuppressin were dose-dependent (10−9 to 10−6 mol l−1 tested), with threshold concentrations (10−8−10−7 mol l−1) consistent with the peptide serving as a circulating hormone. Although cycle frequency, a parameter directly determined by the CG, consistently decreased when pQDLDHVFLRFamide was applied to all preparation types, the magnitudes of this decrease differed, suggesting the possibility that, because myosuppressin modulates the CG and the periphery, it also alters peripheral feedback to the CG. PMID:19946074

  11. Validation of family cancer history data in high-risk families: the influence of cancer site, ethnicity, kinship degree, and multiple family reporters.

    PubMed

    Tehranifar, Parisa; Wu, Hui-Chen; Shriver, Tom; Cloud, Ann J; Terry, Mary Beth

    2015-02-01

    Information on family cancer history (FCH) is often collected for first-degree relatives, but more extensive FCH information is critical for greater accuracy in risk assessment. Using self-reported diagnosis of cancer as the gold standard, we examined differences in the sensitivity and specificity of relative-reported FCH by cancer site, race/ethnicity, language preference, and kinship degree (1,524 individuals from 557 families; average number of relatives per family = 2.7). We evaluated the impact of FCH data collected in 2007-2013 from multiple relatives by comparing mean values and proportions for the number of relatives with any cancer, breast cancer, or ovarian cancer as reported by a single relative and by multiple relatives in the same family. The sensitivity of FCH was lower in Hispanics, Spanish-speaking persons, and third-degree relatives (e.g., for all cancers, sensitivities were 80.7%, 87.4%, and 91.0% for third-, second-, and first-degree relatives, respectively). FCH reported by multiple relatives included a higher number of relatives with cancer than the number reported by a single relative (e.g., mean increase of 1.2 relatives with any cancer), with more relatives diagnosed with any cancer, breast cancer, and ovarian cancer in 52%, 36% and 12% of families, respectively. Collection of FCH data from multiple relatives may provide a more comprehensive picture of FCH and may potentially improve risk assessment and preventive care. © The Author 2015. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. MODEL IMPLEMENTATION TO EVALUATE THE COLLECTIVE FUTURE RADIONUCLIDE RELEASES FROM MULTIPLE FACILITIES AT THE SAVANNAH RIVER SITE

    SciTech Connect

    Hiergesell, R.; Smith, F.; Hamm, L.; Phifer, M.; Swingle, R.

    2009-12-15

    A comprehensive Composite Analysis (CA) has been performed considering 152 sources of residual radioactive material at the Savannah River Site (SRS). As part of the CA a model was developed to perform deterministic base case calculations using the commercial GoldSim software. The model treated transport and decay of radionuclides as they are released at the source location and transported through the source region, vadose zone and aquifer to stream outcrops and from there to the Savannah River. A dose to the public was calculated assuming recreational use of stream water and residential use of river water. The specific results from the GoldSim modeling evaluation conducted as part of the CA indicate that the collective maximum dose resulting from the release of radionuclides from all 152 anticipated SRS End State sources of residual radionuclides demonstrate that maximum exposures expected to occur to any offsite MOP will not approach the 300 uSv/yr (30 mrem/yr) dose constraint, and in fact are currently estimated to be only 10% of this. For each of the POA's evaluated, the highest cumulative dose is realized at the Lower Three Runs POA and is calculated to be 29.7 uSv/yr (2.97 mrem/yr). The major dose contributing radionuclide for all of the POA's, with the exception of Upper Three Runs, was {sup 137}Cs in the contaminated streambed sediments. In Upper Three Runs {sup 237}Np from the H-Area Canyon Building was the major dose contributing radionuclide. The major exposure pathway for the SRS streams (where the Recreational Scenario was evaluated) was by the ingestion of fish. In the Savannah River, where the Residential Scenario was evaluated, ingestion of vegetation was the dominant exposure pathway. The uncertainty evaluation lends added assurance to the conclusion that the 30 mrem/yr dose constraint will not be exceeded, in that even at the 95th Percentile, this performance measure is not expected to be exceeded. It must also be added that these conclusions

  13. A Hypothetical Scenario for Full-Scale Deployment of Geological Carbon Sequestration: Investigating the Interaction Between Multiple CO2 Storage Sites in a Sedimentary Basin

    NASA Astrophysics Data System (ADS)

    Birkholzer, J.; Zhou, Q.; Jordan, P.; Tsang, C.; Leetaru, H.; Mehnert, E.; Frailey, S.; Finley, R.

    2008-12-01

    Most ongoing projects of geological carbon sequestration (GCS) are relatively small in size, with annual injection rates from a few thousand to less than a million tonnes. These projects help build the GCS technology with respect to modeling, monitoring, risk assessment, and mitigation, and have been successful so far in terms of CO2 containment and caprock geomechanical integrity. In the future, GCS will be implemented at full-scale, multiple industrial-size CO2 storage sites in large sedimentary basins to make full use of the potential storage capacity. Simultaneous injection into multiple not-too-distant storage sites will lead to interference between the individual regions of pressure build-up and possible interference between the individual CO2 plumes. The Illinois Basin is used to model the future impact of multiple injection sites in the thick, extensive Mount Simon Formation. The basin-scale model domain of 241,000 km2 covers a core injection area of 24,000 km2, a larger near-field area where significant pressure buildup is expected, and an even larger far-field area for investigating environmental impacts on groundwater resources. The model assumes that there are twenty sequestration sites (spaced 30 km apart) within the core injection area. Three injection scenarios are considered, featuring annual injection rates of 5, 10, and 15 million tonnes of CO2 at each site, respectively. These scenarios correspond to 33%, 67% and 100% of the current single-point large CO2 sources in the relevant states (Illinois, Indiana and Kentucky). The model adequately captures the characteristics of the Mount Simon Formation in the core injection area, which include (1) an overall thickness of 300 to 680 m, (2) an upper unit of sandstone and shale tidally influenced and deposited, (3) a thick middle unit of clean sandstone of relatively high permeability, and (4) a lower arkosic unit of higher permeability (one Darcy) with an average thickness of 90 m. At each site, CO2 is

  14. Hydraulic characterization of volcanic rocks in Pahute Mesa using an integrated analysis of 16 multiple-well aquifer tests, Nevada National Security Site, 2009–14

    USGS Publications Warehouse

    Garcia, C. Amanda; Jackson, Tracie R.; Halford, Keith J.; Sweetkind, Donald S.; Damar, Nancy A.; Fenelon, Joseph M.; Reiner, Steven R.

    2017-01-20

    An improved understanding of groundwater flow and radionuclide migration downgradient from underground nuclear-testing areas at Pahute Mesa, Nevada National Security Site, requires accurate subsurface hydraulic characterization. To improve conceptual models of flow and transport in the complex hydrogeologic system beneath Pahute Mesa, the U.S. Geological Survey characterized bulk hydraulic properties of volcanic rocks using an integrated analysis of 16 multiple-well aquifer tests. Single-well aquifer-test analyses provided transmissivity estimates at pumped wells. Transmissivity estimates ranged from less than 1 to about 100,000 square feet per day in Pahute Mesa and the vicinity. Drawdown from multiple-well aquifer testing was estimated and distinguished from natural fluctuations in more than 200 pumping and observation wells using analytical water-level models. Drawdown was detected at distances greater than 3 miles from pumping wells and propagated across hydrostratigraphic units and major structures, indicating that neither faults nor structural blocks noticeably impede or divert groundwater flow in the study area.Consistent hydraulic properties were estimated by simultaneously interpreting drawdown from the 16 multiple-well aquifer tests with an integrated groundwater-flow model composed of 11 well-site models—1 for each aquifer test site. Hydraulic properties were distributed across volcanic rocks with the Phase II Pahute Mesa-Oasis Valley Hydrostratigraphic Framework Model. Estimated hydraulic-conductivity distributions spanned more than two orders of magnitude in hydrostratigraphic units. Overlapping hydraulic conductivity ranges among units indicated that most Phase II Hydrostratigraphic Framework Model units were not hydraulically distinct. Simulated total transmissivity ranged from 1,600 to 68,000 square feet per day for all pumping wells analyzed. High-transmissivity zones exceeding 10,000 square feet per day exist near caldera margins and extend

  15. Using biological data from field studies with multiple reference sites as a basis for environmental management: the risks for false positives and false negatives.

    PubMed

    Hanson, Niklas

    2011-03-01

    Field surveys of biological responses can provide valuable information about environmental status and anthropogenic stress. However, it is quite usual for biological variables to differ between sites or change between two periods of time also in the absence of an impact. This means that there is an obvious risk that natural variation will be interpreted as environmental impact, or that relevant effects will be missed due to insufficient statistical power. Furthermore, statistical methods tend to focus on the risks for Type-I error, i.e. false positives. For environmental management, the risk for false negatives is (at least) equally important. The aim of the present study was to investigate how the probabilities for false positives and negatives are affected by experimental set up (number of reference sites and samples per site), decision criteria (statistical method and α-level) and effect size. A model was constructed to simulate data from multiple reference sites, a negative control and a positive control. The negative control was taken from the same distribution as the reference sites and the positive control was just outside the normal range. Using the model, the probabilities to get false positives and false negatives were calculated when a conventional statistical test, based on a null hypothesis of no difference, was used along with alternative tests that were based on the normal range of natural variation. Here, it is tested if an investigated site is significantly inside (equivalence test) and significantly outside (interval test) the normal range. Furthermore, it was tested how the risks for false positives and false negatives are affected by changes in α-level and effect size. The results of the present study show that the strategy that best balances the risks between false positives and false negatives is to use the equivalence test. Besides tests with tabulated p-values, estimates generated using a bootstrap routine were included in the present

  16. SeqSIMLA2_exact: simulate multiple disease sites in large pedigrees with given disease status for diseases with low prevalence.

    PubMed

    Yao, Po-Ju; Chung, Ren-Hua

    2016-02-15

    It is difficult for current simulation tools to simulate sequence data in a pre-specified pedigree structure and pre-specified affection status. Previously, we developed a flexible tool, SeqSIMLA2, for simulating sequence data in either unrelated case-control or family samples with different disease and quantitative trait models. Here we extended the tool to efficiently simulate sequences with multiple disease sites in large pedigrees with a given disease status for each pedigree member, assuming that the disease prevalence is low. SeqSIMLA2_exact is implemented with C++ and is available at http://seqsimla.sourceforge.net. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Multiple myeloma risk variant at 7p15.3 creates an IRF4-binding site and interferes with CDCA7L expression

    PubMed Central

    Li, Ni; Johnson, David C.; Weinhold, Niels; Studd, James B.; Orlando, Giulia; Mirabella, Fabio; Mitchell, Jonathan S.; Meissner, Tobias; Kaiser, Martin; Goldschmidt, Hartmut; Hemminki, Kari; Morgan, Gareth J.; Houlston, Richard S.

    2016-01-01

    Genome-wide association studies have identified several risk loci for multiple myeloma (MM); however, the mechanisms by which they influence MM are unknown. Here by using genetic association data and functional characterization, we demonstrate that rs4487645 G>T, the most highly associated variant (P = 5.30 × 10−25), resides in an enhancer element 47 kb upstream of the transcription start site of c-Myc-interacting CDCA7L. The G-risk allele, associated with increased CDCA7L expression (P=1.95 × 10−36), increases IRF4 binding and the enhancer interacts with the CDCA7L promoter. We show that suppression of CDCA7L limits MM proliferation through apoptosis, and increased CDCA7L expression is associated with adverse patient survival. These findings implicate IRF4-mediated CDCA7L expression in MM biology and indicate how germline variation might confer susceptibility to MM. PMID:27882933

  18. Assay of impure plutonium oxide with the large neutron multiplicity counter for IAEA verification of excess weapons material at the Rocky Flats Environmental Technology Site

    SciTech Connect

    Langner, D.G.; Franco, J.B.; Larsen, R.K.

    1997-11-01

    The large neutron multiplicity counter (LNMC), also known as the 30-gal.-drum neutron multiplicity counter, has now been used successfully by the International Atomic Energy Agency (IAEA) for the Initial Physical Inventory Verification (IPIV) and the first annual Physical Inventory Verification (PIV) of excess weapons plutonium oxide at the Rocky Flats Environmental Technology Site (RFETS). These excess plutonium oxide materials contain a variety of impurities. They are stored two cans to a 10-gal.-drum. The drums contain from 1.3 to 4.0 kg of plutonium. The isotopic declarations vary from can to can but the material averages 6% {sup 240}Pu. During the IPIV, 94 samples were measured in the LNMC; 19 were measured during the PIV. The assays for all but a single drum agreed to within three standard deviations of the declared value. This problematic drum could not be measured by the LNMC because of its unusually high neutron emission rate. In this paper we will report on the overall performance of the LNMC in these inspections.

  19. A Novel System for Simultaneous or Sequential Integration of Multiple Gene-Loading Vectors into a Defined Site of a Human Artificial Chromosome

    PubMed Central

    Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

    2014-01-01

    Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming. PMID:25303219

  20. Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector

    PubMed Central

    Monk, Ian R; Casey, Pat G; Cronin, Michael; Gahan, Cormac GM; Hill, Colin

    2008-01-01

    Background The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays. Results We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of L. monocytogenes from complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of L. monocytogenes commonly used in laboratory studies. Conclusion This study has developed a competitive index assay that can be broadly applied to all L. monocytogenes strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of L. monocytogenes in complex biological samples. PMID:18554399

  1. Using radial basis function on the general form of Chou's pseudo amino acid composition and PSSM to predict subcellular locations of proteins with both single and multiple sites.

    PubMed

    Huang, Chao; Yuan, Jingqi

    2013-07-01

    Prediction of protein subcellular location is a meaningful task which attracted much attention in recent years. A lot of protein subcellular location predictors which can only deal with the single-location proteins were developed. However, some proteins may belong to two or even more subcellular locations. It is important to develop predictors which will be able to deal with multiplex proteins, because these proteins have extremely useful implication in both basic biological research and drug discovery. Considering the circumstance that the number of methods dealing with multiplex proteins is limited, it is meaningful to explore some new methods which can predict subcellular location of proteins with both single and multiple sites. Different methods of feature extraction and different models of predict algorithms using on different benchmark datasets may receive some general results. In this paper, two different feature extraction methods and two different models of neural networks were performed on three benchmark datasets of different kinds of proteins, i.e. datasets constructed specially for Gram-positive bacterial proteins, plant proteins and virus proteins. These benchmark datasets have different number of location sites. The application result shows that RBF neural network has apparently superiorities against BP neural network on these datasets no matter which type of feature extraction is chosen.

  2. Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

    PubMed

    Friedrich, Michael G; Hancock, Sarah E; Raftery, Mark J; Truscott, Roger J W

    2016-08-12

    Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic.Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS.

  3. Multiple scattering of waves in random media: Application to the study of the city-site effect in Mexico City area.

    NASA Astrophysics Data System (ADS)

    Ishizawa, O. A.; Clouteau, D.

    2007-12-01

    Long-duration, amplifications and spatial response's variability of the seismic records registered in Mexico City during the September 1985 earthquake cannot only be explained by the soil velocity model. We will try to explain these phenomena by studying the extent of the effect of buildings' diffracted wave fields during an earthquake. The main question is whether the presence of a large number of buildings can significantly modify the seismic wave field. We are interested in the interaction between the incident wave field propagating in a stratified half- space and a large number of structures at the free surface, i.e., the coupled city-site effect. We study and characterize the seismic wave propagation regimes in a city using the theory of wave propagation in random media. In the coupled city-site system, the buildings are modeled as resonant scatterers uniformly distributed at the surface of a deterministic, horizontally layered elastic half-space representing the soil. Based on the mean-field and the field correlation equations, we build a theoretical model which takes into account the multiple scattering of seismic waves and allows us to describe the coupled city-site system behavior in a simple and rapid way. The results obtained for the configurationally averaged field quantities are validated by means of 3D results for the seismic response of a deterministic model. The numerical simulations of this model are computed with MISS3D code based on classical Soil-Structure Interaction techniques and on a variational coupling between Boundary Integral Equations for a layered soil and a modal Finite Element approach for the buildings. This work proposes a detailed numerical and a theoretical analysis of the city-site interaction (CSI) in Mexico City area. The principal parameters in the study of the CSI are the buildings resonant frequency distribution, the soil characteristics of the site, the urban density and position of the buildings in the city, as well as

  4. Frequency tuning of the cervical vestibular-evoked myogenic potential (cVEMP) recorded from multiple sites along the sternocleidomastoid muscle in normal human subjects.

    PubMed

    Wei, Wei; Jeffcoat, Ben; Mustain, William; Zhu, Hong; Eby, Thomas; Zhou, Wu

    2013-02-01

    Frequency tuning of tone burst-evoked myogenic potentials recorded from the sternocleidomastoid muscle (cervical VEMP or cVEMP) is used clinically to assess vestibular function. Understanding the characteristics of cVEMP is important for improving the specificity of cVEMP testing in diagnosing vestibular deficits. In the present study, we analyzed the frequency tuning properties of the cVEMPs by constructing detailed tuning curves and examining their morphology and dependence on SCM tonic level, sound intensity, and recording site along the SCM. Here we report two main findings. First, by employing nine tone frequencies between 125 and 4,000 Hz, some tuning curves exhibited two distinct peaks, which cannot be modeled by a single mass spring system as previously suggested. Instead, the observed tuning is better modeled as linear summation of two mass spring systems, with resonance frequencies at ~300 and ~1,000 Hz. Peak frequency of cVEMP tuning curves was not affected by SCM tonic level, sound intensity, and location of recording site on the SCM. However, sharpness of cVEMP tuning was increased at lower sound intensities. Second, polarity of cVEMP responses recorded from the lower quarter of the SCM was reversed as compared to that at the two upper sites. While more studies are needed, these results suggest that cVEMP tuning is mediated through multiple generators with different resonance frequencies. Future studies are needed to explore implications of these results on development of selective VEMP tests and determine the nature of polarity inversion at the lower quarter of SCM.

  5. Neurogenesis is enhanced by stroke in multiple new stem cell niches along the ventricular system at sites of high BBB permeability.

    PubMed

    Lin, Ruihe; Cai, Jingli; Nathan, Cody; Wei, Xiaotao; Schleidt, Stephanie; Rosenwasser, Robert; Iacovitti, Lorraine

    2015-02-01

    Previous studies have established the subventricular (SVZ) and subgranular (SGZ) zones as sites of neurogenesis in the adult forebrain (Doetsch et al., 1999a; Doetsch, 2003a). Work from our laboratory further indicated that midline structures known as circumventricular organs (CVOs) also serve as adult neural stem cell (NSC) niches (Bennett et al., 2009, 2010). In the quiescent rat brain, NSC proliferation remains low in all of these sites. Therefore, we recently examined whether ischemic stroke injury (MCAO) or sustained intraventricular infusion of the mitogen bFGF could trigger an up-regulation in NSC proliferation, inducing neurogenesis and gliogenesis. Our data show that both stroke and bFGF induce a dramatic and long-lasting (14day) rise in the proliferation (BrdU+) of nestin+Sox2+GFAP+ NSCs capable of differentiating into Olig2+ glial progenitors, GFAP+nestin-astrocyte progenitors and Dcx+ neurons in the SVZ and CVOs. Moreover, because of the upsurge in NSC number, it was possible to detect for the first time several novel stem cell niches along the third (3V) and fourth (4V) ventricles. Importantly, a common feature of all brain niches was a rich vasculature with a blood-brain-barrier (BBB) that was highly permeable to systemically injected sodium fluorescein. These data indicate that stem cell niches are more extensive than once believed and exist at multiple sites along the entire ventricular system, consistent with the potential for widespread neurogenesis and gliogenesis in the adult brain, particularly after injury. We further suggest that because of their leaky BBB, stem cell niches are well-positioned to respond to systemic injury-related cues which may be important for stem-cell mediated brain repair. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Diversity of Enterococcus faecalis Genotypes from Multiple Oral Sites Associated with Endodontic Failure Using Repetitive Sequence-based Polymerase Chain Reaction and Arbitrarily Primed Polymerase Chain Reaction.

    PubMed

    Delboni, Maraísa G; Gomes, Brenda P F A; Francisco, Priscila A; Teixeira, Fabrício B; Drake, David

    2017-03-01

    The aim of this study was to evaluate the diversity and similarity of Enterococcus faecalis genotype isolates from multiple oral sites using repetitive sequence-based polymerase chain reaction and arbitrarily primed polymerase chain reaction (AP-PCR). Forty-two endodontically treated teeth with apical periodontitis were selected. A total of 126 microbial samples were collected from 3 different sites (saliva, pulp chamber, and root canals, all n = 42) during the nonsurgical retreatment procedures. After growth on m-Enterococcus agar, the colonies were isolated, characterized as gram-positive catalase negative cocci, and identified using an API 20 Strep kit (bioMérieux, Marcy-l'Etoile, France). Seventy-four colonies from 10 patients were confirmed as E. faecalis by polymerase chain reaction (16S ribosomal RNA). Repetitive sequence-based polymerase chain reactions using ERIC and AP-PCR using RW3A primers were performed in all 74 colonies. Fingerprints were analyzed and separated into genotypic groups based on the Dice coefficient percentage of similarity (82% or greater) as determined by ERIC reproducibility assays involving E. faecalis controls. Seven different E. faecalis genotypes (GTs) (GT1 = 27%, GT2 = 17.6%, GT3 = 1.3%, GT4 = 18.9%, GT5 = 9.5%, GT6 = 14.9%, and GT7 = 10.8%) were observed in different subjects and oral sites associated with endodontic failure. Remarkably, in 4 of 5 patients, the same GTs present in the infected root canals were also isolated from either the pulp chamber or the saliva samples. In particular, GT6 was detected in all 3 oral sites of patient 37. E. faecalis GTs isolated from saliva, the pulp chamber, and the root canal were similar using the Rep-PCR and AP-PCR methods. These findings suggest that coronal microleakage is a conceivable cause of endodontic failure. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  7. Holocene climate variability in the European Arctic reconstructed from multiple proxy indicators and sites simultaneously using Bayesian multi-scale smoothing (Invited)

    NASA Astrophysics Data System (ADS)

    Korhola, A.; Weckström, J.; Erästö, P.; Holmström, L.

    2009-12-01

    We report a method for reconstructing temperatures from proxy data based on Bayesian multi-scale smoothing. The main features of the method is that it makes possible a simultaneous utilization of multiple proxies and sites to achieve a regional ‘consensus’ reconstruction of temperature, it enables various types of uncertainties (e.g., chronology) to be taken into account, and that it also allows significant features to be detected from the emerging time series. Applying the method to multiple proxy data derived from lakes in Finnish Lapland suggests that, contrary to the conventional view, Holocene climate cooling in the North Atlantic region started already c. 8.5 kyr, and that climate since then was punctuated by several quasi-cyclical climate events, the forcing mechanisms of which are not yet fully understood. Our study suggests that inconsistencies in climate reconstructions between different proxy sources may be at least partly spurious; there is probably no single narrative that counts as the canonical version of Holocene climate change. Instead, there may exist many ’truths’ depending on the proxy and the resolution the data are gained and examined. We expect this method to be effective for generating, synthesizing and interpreting paleoclimate records. BSiZer interpretation of the multi-proxy temperature reconstruction over the Holocene for northern Fennoscandia. Significant upward (positive) slopes are indicated in red, downward (negative) ones in blue. In the grey areas, no conclusions about the significance (λ=0.8) of the slopes can be drawn. Smoothing levels (log10 λ) are shown on the left.

  8. Assessment of BTEX-induced health risk under multiple uncertainties at a petroleum-contaminated site: An integrated fuzzy stochastic approach

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaodong; Huang, Guo H.

    2011-12-01

    Groundwater pollution has gathered more and more attention in the past decades. Conducting an assessment of groundwater contamination risk is desired to provide sound bases for supporting risk-based management decisions. Therefore, the objective of this study is to develop an integrated fuzzy stochastic approach to evaluate risks of BTEX-contaminated groundwater under multiple uncertainties. It consists of an integrated interval fuzzy subsurface modeling system (IIFMS) and an integrated fuzzy second-order stochastic risk assessment (IFSOSRA) model. The IIFMS is developed based on factorial design, interval analysis, and fuzzy sets approach to predict contaminant concentrations under hybrid uncertainties. Two input parameters (longitudinal dispersivity and porosity) are considered to be uncertain with known fuzzy membership functions, and intrinsic permeability is considered to be an interval number with unknown distribution information. A factorial design is conducted to evaluate interactive effects of the three uncertain factors on the modeling outputs through the developed IIFMS. The IFSOSRA model can systematically quantify variability and uncertainty, as well as their hybrids, presented as fuzzy, stochastic and second-order stochastic parameters in health risk assessment. The developed approach haw been applied to the management of a real-world petroleum-contaminated site within a western Canada context. The results indicate that multiple uncertainties, under a combination of information with various data-quality levels, can be effectively addressed to provide supports in identifying proper remedial efforts. A unique contribution of this research is the development of an integrated fuzzy stochastic approach for handling various forms of uncertainties associated with simulation and risk assessment efforts.

  9. Integrating individual-based indices of contaminant effects. How multiple sublethal effects may ultimately reduce amphibian recruitment from a contaminated breeding site.

    PubMed

    Rowe, C L; Hopkins, W A; Congdon, J D

    2001-11-29

    Habitat contamination can alter numerous biological processes in individual organisms. Examining multiple individual-level responses in an integrative fashion is necessary to understand how individual health or fitness reflects environmental contamination. Here we provide an example of such an integrated perspective based upon recent studies of an amphibian (the bullfrog, Rana catesbeiana ) that experiences several, disparate changes when larval development occurs in a trace element-contaminated habitat. First, we present an overview of studies focused on specific responses of individuals collected from, or transplanted into, a habitat contaminated by coal combustion residues (CCR). These studies have reported morphological, behavioral, and physiological modifications to individuals chronically interacting with sediments in the CCR-contaminated site. Morphological abnormalities in the oral and tail regions in contaminant-exposed individuals influenced other properties such as grazing, growth, and swimming performance. Behavioral changes in swimming activities and responses to stimuli appear to influence predation risk in the contaminant-exposed population. Significant changes in bioenergetics in the contaminated habitat, evident as abnormally high energetic expenditures for survival (maintenance) costs, may ultimately influence production pathways (growth, energy storage) in individuals. We then present a conceptual model to examine how interactions among the affected systems (morphological, behavioral, physiological) may ultimately bring about more severe effects than would be predicted if the responses were considered in isolation. A complex interplay among simultaneously occurring biological changes emerges in which multiple, sublethal effects ultimately can translate into reductions in larval or juvenile survival, and thus reduced recruitment of juveniles into the population. In systems where individuals are exposed to low concentrations of contaminants for

  10. Distinct non-target site mechanisms endow resistance to glyphosate, ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant Lolium rigidum.

    PubMed

    Yu, Qin; Abdallah, Ibrahim; Han, Heping; Owen, Mechelle; Powles, Stephen

    2009-09-01

    This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [(14)C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.

  11. Temperature Changes in Cortical Bone after Implant Site Preparation Using a Single Bur versus Multiple Drilling Steps: An In Vitro Investigation.

    PubMed

    Gehrke, Sergio Alexandre; Bettach, Raphaél; Taschieri, Silvio; Boukhris, Gilles; Corbella, Stefano; Del Fabbro, Massimo

    2015-08-01

    The study aims to test the hypothesis of no differences in temperature variation by using a single bur for implant site preparation as compared with conventional drilling sequence using multiple burs with incremental diameter. Synthetic blocks of bone (type I density) were used for drilling procedures. Group 1 and Group 2 - drilling with three consecutive burs for a 4.1 mm cylindrical implant and for a 4.3 mm conical implant, respectively; Group 3 - drilling with a single bur for a 4.2 mm conical implant. For each group, 20 drilling procedures were performed without irrigation and 20 with external irrigation. The temperature in the cortical bone during osteotomy for implant site preparation was measured through a thermocouple. The mean temperatures and standard deviations for the drilling without irrigation were: 25.5 ± 1.24°C for Group 1; 28.1 ± 1.76°C for Group 2; 26.5 ± 1.79°C for Group 3. Considering the drilling with irrigation, the mean values and standard deviations were: 20.4 ± 1.17°C for Group 1; 22.2 ± 1.38°C for Group 2; 20.2 ± 0.83°C for Group 3. Groups 1 and 3 yielded similar results, while Group 2 displayed significantly higher temperature increase than the other two groups. The single bur drilling protocol did not produce greater bone heating than the conventional protocol and may be considered a safe procedure. © 2013 Wiley Periodicals, Inc.

  12. The route of HIV escape from immune response targeting multiple sites is determined by the cost-benefit tradeoff of escape mutations.

    PubMed

    Batorsky, Rebecca; Sergeev, Rinat A; Rouzine, Igor M

    2014-10-01

    Cytotoxic T lymphocytes (CTL) are a major factor in the control of HIV replication. CTL arise in acute infection, causing escape mutations to spread rapidly through the population of infected cells. As a result, the virus develops partial resistance to the immune response. The factors controlling the order of mutating epitope sites are currently unknown and would provide a valuable tool for predicting conserved epitopes. In this work, we adapt a well-established mathematical model of HIV evolution under dynamical selection pressure from multiple CTL clones to include partial impairment of CTL recognition, [Formula: see text], as well as cost to viral replication, [Formula: see text]. The process of escape is described in terms of the cost-benefit tradeoff of escape mutations and predicts a trajectory in the cost-benefit plane connecting sequentially escaped sites, which moves from high recognition loss/low fitness cost to low recognition loss/high fitness cost and has a larger slope for early escapes than for late escapes. The slope of the trajectory offers an interpretation of positive correlation between fitness costs and HLA binding impairment to HLA-A molecules and a protective subset of HLA-B molecules that was observed for clinically relevant escape mutations in the Pol gene. We estimate the value of [Formula: see text] from published experimental studies to be in the range (0.01-0.86) and show that the assumption of complete recognition loss ([Formula: see text]) leads to an overestimate of mutation cost. Our analysis offers a consistent interpretation of the commonly observed pattern of escape, in which several escape mutations are observed transiently in an epitope. This non-nested pattern is a combined effect of temporal changes in selection pressure and partial recognition loss. We conclude that partial recognition loss is as important as fitness loss for predicting the order of escapes and, ultimately, for predicting conserved epitopes that can be

  13. Multiple Antigenic Sites Are Involved in Blocking the Interaction of GII.4 Norovirus Capsid with ABH Histo-Blood Group Antigens

    PubMed Central

    Parra, Gabriel I.; Abente, Eugenio J.; Sandoval-Jaime, Carlos; Sosnovtsev, Stanislav V.; Bok, Karin

    2012-01-01

    Noroviruses are major etiological agents of acute viral gastroenteritis. In 2002, a GII.4 variant (Farmington Hills cluster) spread so rapidly in the human population that it predominated worldwide and displaced previous GII.4 strains. We developed and characterized a panel of six monoclonal antibodies (MAbs) directed against the capsid protein of a Farmington Hills-like GII.4 norovirus strain that was associated with a large hospital outbreak in Maryland in 2004. The six MAbs reacted with high titers against homologous virus-like particles (VLPs) by enzyme-linked immunoassay but did not react with denatured capsid protein in immunoblots. The expression and self-assembly of newly developed genogroup I/II chimeric VLPs showed that five MAbs bound to the GII.4 protruding (P) domain of the capsid protein, while one recognized the GII.4 shell (S) domain. Cross-competition assays and mutational analyses showed evidence for at least three distinct antigenic sites in the P domain and one in the S domain. MAbs that mapped to the P domain but not the S domain were able to block the interaction of VLPs with ABH histo-blood group antigens (HBGA), suggesting that multiple antigenic sites of the P domain are involved in HBGA blocking. Further analysis showed that two MAbs mapped to regions of the capsid that had been associated with the emergence of new GII.4 variants. Taken together, our data map antibody and HBGA carbohydrate binding to proximal regions of the norovirus capsid, showing that evolutionary pressures on the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes. PMID:22532688

  14. The Route of HIV Escape from Immune Response Targeting Multiple Sites Is Determined by the Cost-Benefit Tradeoff of Escape Mutations

    PubMed Central

    Batorsky, Rebecca; Sergeev, Rinat A.; Rouzine, Igor M.

    2014-01-01

    Cytotoxic T lymphocytes (CTL) are a major factor in the control of HIV replication. CTL arise in acute infection, causing escape mutations to spread rapidly through the population of infected cells. As a result, the virus develops partial resistance to the immune response. The factors controlling the order of mutating epitope sites are currently unknown and would provide a valuable tool for predicting conserved epitopes. In this work, we adapt a well-established mathematical model of HIV evolution under dynamical selection pressure from multiple CTL clones to include partial impairment of CTL recognition, , as well as cost to viral replication, . The process of escape is described in terms of the cost-benefit tradeoff of escape mutations and predicts a trajectory in the cost-benefit plane connecting sequentially escaped sites, which moves from high recognition loss/low fitness cost to low recognition loss/high fitness cost and has a larger slope for early escapes than for late escapes. The slope of the trajectory offers an interpretation of positive correlation between fitness costs and HLA binding impairment to HLA-A molecules and a protective subset of HLA-B molecules that was observed for clinically relevant escape mutations in the Pol gene. We estimate the value of from published experimental studies to be in the range (0.01–0.86) and show that the assumption of complete recognition loss () leads to an overestimate of mutation cost. Our analysis offers a consistent interpretation of the commonly observed pattern of escape, in which several escape mutations are observed transiently in an epitope. This non-nested pattern is a combined effect of temporal changes in selection pressure and partial recognition loss. We conclude that partial recognition loss is as important as fitness loss for predicting the order of escapes and, ultimately, for predicting conserved epitopes that can be targeted by vaccines. PMID:25356981

  15. Modeling municipal solid waste collection: A generalized vehicle routing model with multiple transfer stations, gather sites and inhomogeneous vehicles in time windows.

    PubMed

    Son, Le Hoang; Louati, Amal

    2016-06-01

    Municipal Solid Waste (MSW) collection is a necessary process in any municipality resulting in the quality-of-life, economic aspects and urban structuralization. The intrinsic nature of MSW collection relates to the development of effective vehicle routing models that optimize the total traveling distances of vehicles, the environmental emission and the investment costs. In this article, we propose a generalized vehicle routing model including multiple transfer stations, gather sites and inhomogeneous vehicles in time windows for MSW collection. It takes into account traveling in one-way routes, the number of vehicles per m(2) and waiting time at traffic stops for reduction of operational time. The proposed model could be used for scenarios having similar node structures and vehicles' characteristics. A case study at Danang city, Vietnam is given to illustrate the applicability of this model. The experimental results have clearly shown that the new model reduces both total traveling distances and operational hours of vehicles in comparison with those of practical scenarios. Optimal routes of vehicles on streets and markets at Danang are given. Those results are significant to practitioners and local policy makers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Multiple recurrent mutations at four human Y-chromosomal single nucleotide polymorphism sites in a 37 bp sequence tract on the ARSDP1 pseudogene.

    PubMed

    Niederstätter, Harald; Berger, Burkhard; Erhart, Daniel; Willuweit, Sascha; Geppert, Maria; Gassner, Christoph; Schennach, Harald; Parson, Walther; Roewer, Lutz

    2013-12-01

    The male-specific region of the human Y chromosome (MSY) is passed down clonally from father to son and mutation is the single driving force for Y-chromosomal diversification. The geographical distribution of MSY variation is non-random. Therefore, Y-chromosomal single nucleotide polymorphisms (Y-SNPs) are of forensic interest, as they can be utilized, e.g. for deducing the bio-geographical origin of biological evidence. This extra information can complement short tandem repeat data in criminal investigations. For forensic applications, however, any targeted marker has to be unequivocally interpretable. Here, we report findings for 17 samples from a population study comprising specimens from ∼3700 men living in Tyrol (Austria), indicating apparent homoplasic mutations at four Y-SNP loci on haplogroup R-M412/L51/S167, R-U152/S28, and L-M20 Y chromosomes. The affected Y-SNPs P41, P37, L202, and L203 mapped to a 37bp region on Yq11.21. Observing in multiple phylogenetic contexts up to four homoplasic mutations within such a short sequence tract is unlikely to result from a series of independent parallel mutations. Hence, we rather propose X-to-Y gene conversion as a more likely scenario. Practical implications arising from markers exhibiting paralogues on the Y chromosome or sites with a high propensity to recurrent mutation for database searches are addressed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Detection and Site Localization of Phosphorylcholine-Modified Peptides by NanoLC-ESI-MS/MS Using Precursor Ion Scanning and Multiple Reaction Monitoring Experiments

    NASA Astrophysics Data System (ADS)

    Timm, Thomas; Lenz, Christof; Merkel, Dietrich; Sadiffo, Christian; Grabitzki, Julia; Klein, Jochen; Lochnit, Guenter

    2015-03-01

    Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline ( m/z 104.1) and phosphorylcholine ( m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.

  18. Detection and site localization of phosphorylcholine-modified peptides by NanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring experiments.

    PubMed

    Timm, Thomas; Lenz, Christof; Merkel, Dietrich; Sadiffo, Christian; Grabitzki, Julia; Klein, Jochen; Lochnit, Guenter

    2015-03-01

    Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline (m/z 104.1) and phosphorylcholine (m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.

  19. Characterisation of multiple regulatory domains spanning the major transcriptional start site of the FUS gene, a candidate gene for motor neurone disease.

    PubMed

    Khursheed, Kejhal; Wilm, Thomas P; Cashman, Christine; Quinn, John P; Bubb, Vivien J; Moss, Diana J

    2015-01-21

    Fused-In-Sarcoma (FUS) is a candidate gene for neurological disorders including motor neurone disease and Parkinson׳s disease in addition to various types of cancer. Recently it has been reported that over expression of FUS causes motor neurone disease in mouse models hence mutations leading to changes in gene expression may contribute to the development of neurodegenerative disease. Genome evolutionary conservation was used to predict important cis-acting DNA regulators of the FUS gene promoter that direct transcription. The putative regulators identified were analysed in reporter gene assays in cells and in chick embryos. Our analysis indicated in addition to regulatory domains 5' of the transcriptional start site an important regulatory domain resides in intron 1 of the gene itself. This intronic domain functioned both in cell lines and in vivo in the neural tube of the chick embryo including developing motor neurones. Our data suggest the interaction of multiple domains including intronic domains are involved in expression of FUS. A better understanding of the regulation of expression of FUS may give insight into how its stimulus inducible expression may be associated with neurological disorders.

  20. Designing Allosteric Regulators of Thrombin. Exosite 2 Features Multiple Sub-Sites That Can Be Targeted By Sulfated Small Molecules for Inducing Inhibition

    PubMed Central

    Sidhu, Preetpal Singh; Abdel Aziz, May H.; Sarkar, Aurijit; Mehta, Akul Y.; Zhou, Qibing; Desai, Umesh R.

    2013-01-01

    We recently designed a group of novel exosite 2-directed, sulfated, small, allosteric inhibitors of thrombin. To develop more potent inhibitors, monosulfated benzofuran tri- and tetrameric homologs of the parent designed dimers were synthesized in 7–8 steps and found to exhibit a wide range of potencies. Among these, trimer 9a was found to be nearly 10-fold more potent than the first generation molecules. Michaelis-Menten studies indicated an allosteric mechanism of inhibition. Competitive studies using a hirudin peptide (exosite 1 ligand) and, unfractionated heparin, heparin octasaccharide and γ′-fibrinogen peptide (exosite 2 ligands), demonstrated exosite 2 recognition in a manner different from the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a potency for Arg233Ala thrombin only confirming the major difference in site of recognition between the two structurally related sulfated benzofurans. The results suggest that multiple avenues are available within exosite 2 for inducing thrombin inhibition. PMID:23718540

  1. On the use of multiple-point statistics to improve groundwater flow modeling in karst aquifers: A case study from the Hydrogeological Experimental Site of Poitiers, France

    NASA Astrophysics Data System (ADS)

    Le Coz, Mathieu; Bodin, Jacques; Renard, Philippe

    2017-02-01

    Limestone aquifers often exhibit complex groundwater flow behaviors resulting from depositional heterogeneities and post-lithification fracturing and karstification. In this study, multiple-point statistics (MPS) was applied to reproduce karst features and to improve groundwater flow modeling. For this purpose, MPS realizations were used in a numerical flow model to simulate the responses to pumping test experiments observed at the Hydrogeological Experimental Site of Poitiers, France. The main flow behaviors evident in the field data were simulated, particularly (i) the early-time inflection of the drawdown signal at certain observation wells and (ii) the convex behavior of the drawdown curves at intermediate times. In addition, it was shown that the spatial structure of the karst features at various scales is critical with regard to the propagation of the depletion wave induced by pumping. Indeed, (i) the spatial shape of the cone of depression is significantly affected by the karst proportion in the vicinity of the pumping well, and (ii) early-time inflection of the drawdown signal occurs only at observation wells crossing locally well-developed karst features.

  2. Pseudo-acetylation of multiple sites on human Tau proteins alters Tau phosphorylation and microtubule binding, and ameliorates amyloid beta toxicity.

    PubMed

    Gorsky, Marianna Karina; Burnouf, Sylvie; Sofola-Adesakin, Oyinkan; Dols, Jacqueline; Augustin, Hrvoje; Weigelt, Carina Marianne; Grönke, Sebastian; Partridge, Linda

    2017-08-30

    Tau is a microtubule-associated protein that is highly soluble and natively unfolded. Its dysfunction is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD), where it aggregates within neurons. Deciphering the physiological and pathogenic roles of human Tau (hTau) is crucial to further understand the mechanisms leading to its dysfunction in vivo. We have used a knock-out/knock-in strategy in Drosophila to generate a strain with hTau inserted into the endogenous fly tau locus and expressed under the control of the endogenous fly tau promoter, thus avoiding potential toxicity due to genetic over-expression. hTau knock-in (KI) proteins were expressed at normal, endogenous levels, bound to fly microtubules and were post-translationally modified, hence displaying physiological properties. We used this new model to investigate the effects of acetylation on hTau toxicity in vivo. The simultaneous pseudo-acetylation of hTau at lysines 163, 280, 281 and 369 drastically decreased hTau phosphorylation and significantly reduced its binding to microtubules in vivo. These molecular alterations were associated with ameliorated amyloid beta toxicity. Our results indicate acetylation of hTau on multiple sites regulates its biology and ameliorates amyloid beta toxicity in vivo.

  3. 3D local structure around copper site of rabbit prion-related protein: Quantitative determination by XANES spectroscopy combined with multiple-scattering calculations

    NASA Astrophysics Data System (ADS)

    Cui, P. X.; Lian, F. L.; Wang, Y.; Wen, Yi; Chu, W. S.; Zhao, H. F.; Zhang, S.; Li, J.; Lin, D. H.; Wu, Z. Y.

    2014-02-01

    Prion-related protein (PrP), a cell-surface copper-binding glycoprotein, is considered to be responsible for a number of transmissible spongiform encephalopathies (TSEs). The structural conversion of PrP from the normal cellular isoform (PrPC) to the post-translationally modified form (PrPSc) is thought to be relevant to Cu2+ binding to histidine residues. Rabbits are one of the few mammalian species that appear to be resistant to TSEs, because of the structural characteristics of the rabbit prion protein (RaPrPC) itself. Here we determined the three-dimensional local structure around the C-terminal high-affinity copper-binding sites using X-ray absorption near-edge structure combined with ab initio calculations in the framework of the multiple-scattering (MS) theory. Result shows that two amino acid resides, Gln97 and Met108, and two histidine residues, His95 and His110, are involved in binding this copper(II) ion. It might help us understand the roles of copper in prion conformation conversions, and the molecular mechanisms of prion-involved diseases.

  4. Kinetic modeling of the interactions between 4-methylumbelliferone, 1-naphthol, and zidovudine glucuronidation by udp-glucuronosyltransferase 2B7 (UGT2B7) provides evidence for multiple substrate binding and effector sites.

    PubMed

    Uchaipichat, Verawan; Galetin, Aleksandra; Houston, J Brian; Mackenzie, Peter I; Williams, J Andrew; Miners, John O

    2008-10-01

    Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.

  5. The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    PubMed Central

    Guan, Zhanwen; Zhong, Ling; Li, Chunyan; Wu, Wenbi; Yuan, Meijin

    2016-01-01

    ABSTRACT Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate the ac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma, ac54 ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasin d-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected by ac54 deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly. IMPORTANCE Baculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of

  6. Geologic, water-chemistry, and hydrologic data from multiple-well monitoring sites and selected water-supply wells in the Santa Clara Valley, California, 1999-2003

    USGS Publications Warehouse

    Newhouse, M.W.; Hanson, R.T.; Wentworth, C.M.; Everett, Rhett; Williams, C.F.; Tinsley, J.C.; Noce, T.E.; Carkin, B.A.

    2004-01-01

    To better identify the three-dimensional geohydrologic framework of the Santa Clara Valley, lithologic, geologic, geophysical, geomechanical, hydraulic, and water-chemistry data were collected from eight ground-water multiple-well monitoring sites constructed in Santa Clara County, California, as part of a series of cooperative studies between the U.S. Geological Survey and the Santa Clara Valley Water District. The data are being used to update and improve the three-dimensional geohydrologic framework of the basin and to address issues related to water supply, water chemistry, sequence stratigraphy, geology, and geological hazards. This report represents a compilation of data collected from 1999 to 2003, including location and design of the monitoring sites, cone penetrometer borings, geologic logs, lithologic logs, geophysical logs, core analysis, water-chemistry analysis, ground-water-level measurements, and hydraulic and geomechanical properties from wells and core samples. Exploratory cone penetrometer borings taken in the upper 17 to 130 feet at six of the monitoring sites identified the base of Holocene as no deeper than 75 feet in the central confined area and no deeper than 35 feet in the southern unconfined areas of the valley. Generalized lithologic characterization from the monitoring sites indicates about four to six different aquifer units separated by relatively fine-grained units occur within the alluvial deposits shallower than 860 feet deep. Analysis of geophysical logs indicates that coarse-grained units varied in thickness between 10 and 25 feet in the southeastern unconfined area of the valley and between 50 and 200 feet in the south-central and southwestern areas of the valley. Deviations from temperature-gradient logs indicate that the majority of horizontal ground-water flow occurs above a depth of 775 feet in the south central and above 510 feet in the southeastern areas of the valley. Bulk physical properties from more than 1,150 feet of

  7. What factors contribute to the continued low rates of Indigenous status identification in urban general practice? - A mixed-methods multiple site case study.

    PubMed

    Schütze, Heike; Jackson Pulver, Lisa; Harris, Mark

    2017-01-31

    Indigenous peoples experience worse health and die at younger ages than their non-indigenous counterparts. Ethnicity data enables health services to identify inequalities experienced by minority populations and to implement and monitor services specifically targeting them. Despite significant Government intervention, Australia's Indigenous peoples, the Aboriginal and Torres Strait Islander peoples, continue to be under identified in data sets. We explored the barriers to Indigenous status identification in urban general practice in two areas in Sydney. A mixed-methods multiple-site case study was used, set in urban general practice. Data collection included semi-structured interviews and self-complete questionnaires with 31 general practice staff and practitioners, interviews with three Medicare Local staff, and focus groups with the two local Aboriginal and Torres Strait Islander communities in the study areas. These data were combined with clinical record audit data and Aboriginal unannounced standardised patient visits to participating practices to determine the current barriers to Indigenous status identification in urban general practice. Findings can be broadly grouped into three themes: a lack of practitioner/staff understanding on the need to identify Indigenous status or that a problem with identification exists; suboptimal practice systems to identify and/or record patients' Indigenous status; and practice environments that do not promote Indigenous status identification. Aboriginal and Torres Strait Islander peoples remain under-identified in general practice. There is a need to address the lack of practitioner and staff recognition that a problem with Indigenous status identification exists, along with entrenched attitudes and beliefs and limitations to practice software capabilities. Guidelines recommending Indigenous status identification and Aboriginal and Torres Strait Islander-specific Practice Incentive Payments have had limited impact on

  8. Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing

    PubMed Central

    Hodgson, Jeffrey J.; Arif, Basil M.

    2013-01-01

    The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme. PMID:23302896

  9. Bacterial Prostatitis Enhances 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-Induced Cancer at Multiple Sites

    PubMed Central

    Sfanos, Karen S.; Canene-Adams, Kirstie; Hempel, Heidi; Yu, Shu-Han; Simons, Brian W.; Schaeffer, Anthony J.; Schaeffer, Edward M.; Nelson, William G.; De Marzo, Angelo M.

    2015-01-01

    Dietary carcinogens, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and chronic inflammation have each been implicated as etiological agents in prostate cancer. We hypothesized that bacterial prostatitis would accelerate PhIP-induced pre-invasive lesions in the rat prostate. Male Fischer 344 rats were assigned into 4 groups: Control (untreated), PhIP (200 ppm in the diet for 20 weeks), E. coli (prostatic inoculation in week 10), or PhIP+E. coli. Study animals were monitored for a total of 52 weeks and were euthanized as necessary based on strict criteria for health status and tumor burden. Animals treated with E. coli initially developed acute and chronic inflammation in all lobes of the prostate, whereas inflammation was observed predominantly in the ventral lobe at time of death. PhIP+E. coli-treated animals exhibited a marked decrease in survival compared to PhIP-alone treated animals as a result of an increase in the number of invasive cancers that developed at multiple sites including the skin, small intestine, and Zymbal’s gland. Despite their earlier mortality, PhIP+E. coli-treated animals developed an increased average number of precancerous lesions within the prostate compared to PhIP-treated animals, with a significantly increased Ki-67 index. Multiplexed serum cytokine analysis indicated an increase in the level of circulating IL-6 and IL-12 in PhIP+E. coli-treated animals. Elevated serum IL-6 levels correlated with the development of precancerous lesions within the prostate. These results suggest that bacterial infections and dietary carcinogens - two conceivably preventable cancer risk factors – may synergistically promote tumorigenesis. PMID:25990088

  10. Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale.

    PubMed Central

    Rohr, S; Salzberg, B M

    1994-01-01

    We have applied multiple site optical recording of transmembrane voltage (MSORTV) to patterned growth cultures of heart cells to analyze the effect of geometry per se on impulse propagation in excitable tissue, with cellular and subcellular resolution. Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for this application; it is internalized slowly and permits optical recording with signal-to-noise ratios as high as 40:1 (measured peak-to-peak) and average fractional fluorescence changes of 15% per 100 mV. Using a x 100 objective and a fast data acquisition system, we could resolve impulse propagation on a microscopic scale (15 microns) with high temporal resolution (uncertainty of +/- 5 microseconds). We could observe the decrease in conduction velocity of an impulse propagating along a narrow cell strand as it enters a region of abrupt expansion, and we could explain this phenomenon in terms of the micro-architecture of the tissue. In contrast with the elongated and aligned cells forming the narrow strands, the cells forming the expansions were aligned at random and presented 2.5 times as many cell-to-cell appositions per unit length. If the decrease in conduction velocity results entirely from this increased number of cell-to-cell boundaries per unit length, the mean activation delay introduced by each boundary can be estimated to be 70 microseconds. Using this novel experimental system, we could also demonstrate the electrical coupling of fibroblasts and endotheloid cells to myocytes in culture. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 PMID:7811945

  11. Resting and injury-induced inflamed periosteum contain multiple macrophage subsets that are located at sites of bone growth and regeneration.

    PubMed

    Alexander, Kylie Anne; Raggatt, Liza-Jane; Millard, Susan; Batoon, Lena; Chiu-Ku Wu, Andy; Chang, Ming-Kang; Hume, David Arthur; Pettit, Allison Robyn

    2017-01-01

    Better understanding of bone growth and regeneration mechanisms within periosteal tissues will improve understanding of bone physiology and pathology. Macrophage contributions to bone biology and repair have been established but specific investigation of periosteal macrophages has not been undertaken. We used an immunohistochemistry approach to characterize macrophages in growing murine bone and within activated periosteum induced in a mouse model of bone injury. Osteal tissue macrophages (osteomacs) and resident macrophages were distributed throughout resting periosteum. In tissues collected from 4-week-old mice, osteomacs were observed intimately associated with sites of periosteal diaphyseal and metaphyseal bone dynamics associated with normal growth. This included F4/80(+)Mac-2(-/low) osteomac association with extended tracks of bone formation (modeling) on diphyseal periosteal surfaces. Although this recapitulated endosteal osteomac characteristics, there was subtle variance in the morphology and spatial organization of periosteal modeling-associated osteomacs, which likely reflects the greater structural complexity of periosteum. Osteomacs, resident macrophages and inflammatory macrophages (F4/80(+)Mac-2(hi)) were associated with the complex bone dynamics occurring within the periosteum at the metaphyseal corticalization zone. These three macrophage subsets were also present within activated native periosteum after bone injury across a 9-day time course that spanned the inflammatory through remodeling bone healing phases. This included osteomac association with foci of endochondral ossification within the activated native periosteum. These observations confirm that osteomacs are key components of both osteal tissues, in spite of salient differences between endosteal and periosteal structure and that multiple macrophage subsets are involved in periosteal bone dynamics.

  12. Initiation and growth of multiple-site damage in the riveted lap joint of a curved stiffened fuselage panel: An experimental and analytical study

    NASA Astrophysics Data System (ADS)

    Ahmed, Abubaker Ali

    As part of the structural integrity research of the National Aging Aircraft Research Program, a comprehensive study on multiple-site damage (MSD) initiation and growth in a pristine lap-joint fuselage panel has been conducted. The curved stiffened fuselage panel was tested at the Full-Scale Aircraft Structural Test Evaluation and Research (FASTER) facility located at the Federal Aviation Administration William J. Hughes Technical Center. A strain survey test was conducted to verify proper load application. The panel was then subjected to a fatigue test with constant-amplitude cyclic loading. The applied loading spectrum included underload marker cycles so that crack growth history could be reconstructed from post-test fractographic examinations. Crack formation and growth were monitored via nondestructive and high-magnification visual inspections. Strain gage measurements recorded during the strain survey tests indicated that the inner surface of the skin along the upper rivet row of the lap joint experienced high tensile stresses due to local bending. During the fatigue loading, cracks were detected by eddy-current inspections at multiple rivet holes along the upper rivet row. Through-thickness cracks were detected visually after about 80% of the fatigue life. Once MSD cracks from two adjacent rivet holes linked up, there was a quick deterioration in the structural integrity of the lap joint. The linkup resulted in a 2.87" (72.9-mm) lead fatigue crack that rapidly propagated across 12 rivet holes and crossed over into the next skin bay, at which stage the fatigue test was terminated. A post-fatigue residual strength test was then conducted by loading the panel quasi-statically up to final failure. The panel failed catastrophically when the crack extended instantaneously across three additional bays. Post-test fractographic examinations of the fracture surfaces in the lap joint of the fuselage panel were conducted to characterize subsurface crack initiation and

  13. Clostridial antibody response from injection-site lesions in beef cattle, long-term response to single or multiple doses, and response in newborn beef calves.

    PubMed

    Troxel, T R; Gadberry, M S; Wallace, W T; Kreider, D L; Shockey, J D; Colburn, E A; Widel, P; Nicholson, I

    2001-10-01

    Experiments were conducted to compare clostridial antibody response of beef heifers that do and do not develop injection-site lesions, evaluate long-term antibody response of a single- and multiple-dose toxoid, and evaluate the ability of a clostridial toxoid to elicit an active antibody response in newborn calves. In Exp. 1, 37 weaned heifers were vaccinated (d 0) with a clostridial vaccine (Alpha-7, 2 mL, s.c.). Serum samples were collected on d 0, 28, 56, 84, and 112 to determine clostridial antibody titers. On d 28, heifers were visually inspected and palpated for injection-site lesions. The percentage of heifers that developed lesions was 64.9%. Lesioned heifers had elevated antibody titers for Clostridium chauvoei (CC) on d 28 (P < 0.08) and 84 (P < 0.07) compared with non-lesioned heifers. Clostridium sordellii (CS) and perfringens type D (CPD) antibody titers were greater in lesioned heifers than in non-lesioned heifers on d 28 and 56. In Exp. 2, long-term antibody response of Alpha-7 (A7) and Ultrabac 7 (UB7) was investigated in stocker heifers. The A7 heifers (n = 15) received one 2-mL vaccination (d 0), and the UB7 heifers (n = 15) received a 5-mL vaccination on d 0 and 28. Blood samples were collected on d 0, 28, 56, 84, 112, 140, and 180. Clostridium chauvoei, CPD, and Cl. novyi (CN) antibody titers from the A7 heifers were greater than those from the UB7 heifers on d 28. Due to the second UB7 injection, CC, CS, CN, and Cl. perfringens type C (CPC) antibody titers were greater in UB7 heifers than in A7 heifers on d 56. By d 112, titers were not different, and by d 140 all antibody titers were below detectable levels. In Exp. 3, 58 pregnant, mature, crossbred cows were vaccinated with A7 before calving. At birth, calves were carefully observed to ensure consumption of colostrum. Calves were blocked according to parturition date, and calves in each block were randomly allocated to receive A7 (s.c. at 3 +/- 3 d of age) or remain unvaccinated controls

  14. New 40Ar/39Ar age of the Bishop Tuff from multiple sites and sediment rate calibration for the Matuyama-Brunhes boundary

    USGS Publications Warehouse

    Sarna-Wojcicki, A. M.; Pringle, M.S.; Wijbrans, J.

    2000-01-01

    Precise dating of sanidine from proximal ash flow Bishop Tuff and air fall Bishop pumice and ash, California, can be used to derive an absolute age of the Matuyama Reversed-Brunhes Normal (M-B) paleomagnetic transition, identified stratigraphically close beneath the Bishop Tuff and ash at many sites in the western United States. An average age of 758.9 ?? 1.8 ka, standard error of the mean (SEM), was obtained for individual sanidine crystals or groups of several crystals, determined from ???70 individual analyses of sanidine separates from 11 sample groups obtained at five localities. The basal air fall pumice (757.7 ?? 1.8 ka) and overlying ash flow tuff (762.2 ?? 4.7 ka) from near the source yield essentially the same dates within errors of analysis, suggesting that the two units were emplaced close in time. A date on distal Bishop air fall ash bed at Friant, California, ???100 km to the west of the source area, is younger, 750.1 ?? 4.3 ka, but not significantly different within analytical error (??1 standard deviation). Previous dates of the Bishop Tuff, obtained by others using conventional K-Ar and the fission track method on zircons, ranged from ???650 ka to ???1.0 Ma. The most recent, generally accepted date by the K-Ar method on sanidine was 738 ?? 3 ka. We infer, as others before, that many K-Ar dates on sanidine feldspar are too young owing to incomplete degassing of radiogenic Ar during fusion in the K-Ar technique and that many older K-Ar dates are too old owing to detrital or xenocrystic contamination in the larger samples that are necessary for the technique. The new dates are similar to recent 40Ar/39Ar ages of the Bishop Tuff determined on individual samples by others but are derived from a larger proximal sample population and from multiple analysis of each sample. The results provide a definitive and precise age calibration of this widespread chronostratigraphic marker in the western United States and northeastern Pacific Ocean. We calculated the

  15. Multiple-Site Concerted Proton-Electron Transfer Reactions of Hydrogen-Bonded Phenols are Non-adiabatic and Well Described by Semi-Classical Marcus Theory

    PubMed Central

    Schrauben, Joel N.; Cattaneo, Mauricio; Day, Thomas C.; Tenderholt, Adam L.; Mayer, James M.

    2012-01-01

    Photo-oxidations of hydrogen-bonded phenols using excited state polyarenes are described, to derive fundamental understanding of multiple-site concerted proton-electron transfer reactions (MS-CPET). Experiments have examined phenol-bases having −CPh2NH2, −Py, and −CH2Py groups ortho to the phenol hydroxyl group and tert-butyl groups in the 4,6-positions for stability (HOAr-NH2, HOAr-Py, and HOAr-CH2Py, respectively; Py = pyridyl; Ph = phenyl). The photo-oxidations proceed by intramolecular proton transfer from the phenol to the pendent base concerted with electron transfer to the excited polyarene. For comparison, 2,4,6-tBu3C6H2OH, a phenol without a pendent base and tert-butyl groups in the 2,4,6-positions, has also been examined. Many of these bimolecular reactions are fast, with rate constants near the diffusion limit. Combining the photochemical kCPET values with those from prior thermal stopped-flow kinetic studies gives datasets for the oxidations of HOAr-NH2 and of HOAr-CH2Py that span over 107 in kCPET and nearly 0.9 eV in driving force (ΔGo′). Plots of log(kCPET) vs. ΔGo′ define a single Marcus parabola in each case, each including both excited state anthracenes and ground state aminium radical cations. These two datasets are thus well described by semi-classical Marcus theory, providing a strong validation of the use of this theory for MS-CPET. The parabolas give λCPET ≅ 1.15–1.2 eV and Hab ≅ 20–30 cm−1. These experiments represent the most direct measurements of Hab for MS-CPET reactions to date. Although rate constants are available only up to the diffusion limit, the parabolas clearly peak well below the adiabatic limit of ca. 6 × 1012 s−1. Thus, this is a very clear demonstration that the reactions are non-adiabatic. The non-adiabatic character slows the reactions by a factor of ~45. Results for the oxidation of HOAr-Py, in which the phenol and base are conjugated, and for oxidation of 2,4,6-tBu3C6H2OH, which lacks a base

  16. Multiple-site concerted proton-electron transfer reactions of hydrogen-bonded phenols are nonadiabatic and well described by semiclassical Marcus theory.

    PubMed

    Schrauben, Joel N; Cattaneo, Mauricio; Day, Thomas C; Tenderholt, Adam L; Mayer, James M

    2012-10-10

    Photo-oxidations of hydrogen-bonded phenols using excited-state polyarenes are described to derive fundamental understanding of multiple-site concerted proton-electron transfer reactions (MS-CPET). Experiments have examined phenol bases having -CPh(2)NH(2), -Py, and -CH(2)Py groups ortho to the phenol hydroxyl group and tert-butyl groups in the 4,6-positions for stability (HOAr-NH(2), HOAr-Py, and HOAr-CH(2)Py, respectively; Py = pyridyl; Ph = phenyl). The photo-oxidations proceed by intramolecular proton transfer from the phenol to the pendent base concerted with electron transfer to the excited polyarene. For comparison, 2,4,6-(t)Bu(3)C(6)H(2)OH, a phenol without a pendent base and tert-butyl groups in the 2,4,6-positions, has also been examined. Many of these bimolecular reactions are fast, with rate constants near the diffusion limit. Combining the photochemical k(CPET) values with those from prior thermal stopped-flow kinetic studies gives data sets for the oxidations of HOAr-NH(2) and HOAr-CH(2)Py that span over 10(7) in k(CPET) and nearly 0.9 eV in driving force (ΔG(o)'). Plots of log(k(CPET)) vs ΔG(o)', including both excited-state anthracenes and ground state aminium radical cations, define a single Marcus parabola in each case. These two data sets are thus well described by semiclassical Marcus theory, providing a strong validation of the use of this theory for MS-CPET. The parabolas give λ(CPET) ≅ 1.15-1.2 eV and H(ab) ≅ 20-30 cm(-1). These experiments represent the most direct measurements of H(ab) for MS-CPET reactions to date. Although rate constants are available only up to the diffusion limit, the parabolas clearly peak well below the adiabatic limit of ca. 6 × 10(12) s(-1). Thus, this is a very clear demonstration that the reactions are nonadiabatic. The nonadiabatic character slows the reactions by a factor of ~45. Results for the oxidation of HOAr-Py, in which the phenol and base are conjugated, and for oxidation of 2,4,6-(t)Bu(3)C(6)H(2

  17. Impact of vegetation cover and stand age on scaling carbon fluxes in the upper Midwest: a multiple eddy flux site study

    NASA Astrophysics Data System (ADS)

    Desai, A. R.; Normeets, A.; Bolstad, P. V.; Chen, J.; Cook, B. D.; Curtis, P. S.; Davis, K. J.; Euskirchen, E.; Gough, C.; Martin, J.; Ricciuto, D. M.; Schmid, H. P.; Tang, J.; Su, H.; Vogel, C.; Wang, W.

    2004-12-01

    Eight permanent and three roving eddy flux towers were used to observe the exchange of carbon dioxide between the ecosystem and atmosphere at fourteen different sites in northern Wisconsin and Michigan (USA) during the growing seasons (May-Sept) of 2002 and 2003. These towers were part of the Chequamegon Ecosystem-Atmosphere Study (ChEAS), the University of Michigan Biological Station (UMBS), and the Michigan Technical University. The sites spanned a range of vegetation types typical of the region (northern hardwood, hemlock-hardwood, mixed forest, red pine, jack pine, pine barrens and shrub wetland). The hardwood and red pine sites also spanned a range of forest stand age (young, intermediate, mature and old). All sites experienced roughly similar climate; thus, comparisons among the sites allow for an examination of the impact of heterogeneous vegetation cover and stand age across a regional landscape. Carbon fluxes at different sites generally reacted similarly in response to variability in climate. Results suggest that both cover type and stand age are important variables for modeling and predicting fluxes in this region. These results have implications for developing methods of scaling carbon dioxide fluxes from sites to regions. These results will be contrasted to a flux decomposition at the WLEF tall tower.

  18. Assessing natural attenuation potential at a uranium (U) in situ recovery site (Rosita, TX, USA) using multiple redox-sensitive isotope systems

    NASA Astrophysics Data System (ADS)

    Basu, A.; Brown, S. T.; Christensen, J. N.; DePaolo, D. J.; Reimus, P. W.; Heikoop, J. M.; Simmons, A. M.; House, B.; Schilling, K.; Johnson, T. M.; Pelizza, M.

    2013-12-01

    The In Situ Recovery (ISR) U mining operation at Rosita, TX, USA, involved oxidative dissolution of U from roll front U deposits. This process mobilized U along with other characteristic elements (e.g., Se) from the roll fronts in their soluble and toxic oxidized forms (e.g., U(VI), Se(VI)). The dissolved U(VI) in groundwater poses significant ecological risk due to its chemical toxicity and must be restored below the existing regulatory limit to minimize the environmental impact of ISR mining. However, the undisturbed sediments downgradient to the roll front deposits are expected to remain reduced. Naturally occurring Fe-minerals (e.g., FeS, siderite, magnetite) and microorganisms in the sediments downgradient to ISR activity can reduce dissolved U(VI) to less toxic and insoluble U(IV) and promote natural attenuation. The reduction of oxyanions of U or Se induces measurable isotopic fractionation that can be used to monitor the natural attenuation by downgradient sediments. Here, we used multiple redox-sensitive isotope systems (U, Se, and S) to detect reducing conditions and natural attenuation of U(VI) at the ISR site. We collected groundwater samples from 26 wells located in the ore body, upgradient and downgradient to the ore body. The δ238U values measured in groundwater samples from 23 wells range from 0.48‰ to -1.66‰ (×0.12‰). A preliminary investigation of 6 groundwater samples shows a variation of δ82Se values from -1.44‰ to 5.24‰ (×0.15‰). The δ34SO4 measurements in groundwater vary from 11.8‰ to -19.9‰. The reduction of Se(VI) and SO42- fractionates the lighter isotopes (i.e., 32S and 76Se) in the reduced product phase rendering the remaining reactants in the groundwater enriched in heavier isotopes. Therefore, the high δ82Se and δ34SO4 values may suggest reduction of Se(VI) and SO42-, respectively. The highest δ238U values are observed in the wells located in the ore body or upgradient to the ore body whereas the downgradient

  19. Site migration in seeking care services from multiple providers is associated with worse clinical outcomes among HIV-infected individuals in Washington, DC.

    PubMed

    Jia, Yujiang; Sengupta, Debapriya; Opoku, Jenevieve; Wu, Charles; Griffin, Angelique; West, Tiffany; Samala, Rowena; Shaikh, Irshad; Pappas, Gregory

    2014-01-01

    Washington, DC, is a metropolitan city with a severe HIV epidemic and faces challenges in retaining people living with HIV (PLWH) in quality care. This study assessed site migration in seeking care services and its correlates among PLWH in DC. PLWH diagnosed before 2008 and living through the end of 2010 were analyzed. Six scenarios of site migration were examined as patients visited =2, =3, and =4 different providers for their CD4 cell count and/or viral load (VL) tests in the past 3 years from 2008 to 2010 and 2 years from 2009 to 2010, respectively. Of 6480 patients analyzed from 2008 to 2010, 18.4% had CD4 < 200 cells/mm(3), 30.5% had VL > 400 copies/mL, and 76.6% were retained in same care sites; 23.4%, 5.0%, and 0.9% visited =2, =3, and =4 sites in the past 3 years from 2008 to 2010, respectively. Of 5954 patients analyzed from 2009 to 2010, 16.8% had CD4 < 200 cells/mm(3), 29.4% had VL > 400 copies/mL, and 81.9% were retained in same care sites; 18.1%, 3.1%, and 0.6% visited =2, =3, and =4 sites in the past 2 years from 2009 to 2010, respectively. Multivariable logistic regression analyses revealed that migration across six scenarios are consistently associated with CD4 < 200 cells/mm(3) and VL > 400 copies/mL. Site migration was common and associated with lower CD4 and higher VL among PLWH in DC. Frequent migration might be a factor in achieving optimal health outcomes for a subset of patients. Site migration might potentially limit effective delivery of high quality care and treatment services. The preliminary findings underscore the need for further research to assess the predictors of migration and its impact on stage of care.

  20. /sup 125/I-neuropeptide Y and /sup 125/I-peptide YY bind to multiple receptor sites in rat brain

    SciTech Connect

    Walker, M.W.; Miller, R.J.

    1988-12-01

    We describe the preparation of monoiodinated neuropeptide Y (Tyr1-125I-NPY) and monoiodinated peptide YY (Tyr36-125I-PYY). Using these ligands, we detected high, moderate, and low affinity receptor populations in rat brain. Only high and moderate affinity binding sites were suggested by saturation binding studies. Tyr1-125I-NPY bound to 8 +/- 3% of the sites with a Kd of 54 pM (Bmax = 19.4 fmol/mg of protein) and to 92 +/- 3% of the sites with a Kd of 0.92 nM (Bmax = 220.0 fmol/mg of protein). Tyr36-125I-PYY bound to 14 +/- 3% of the sites with a Kd of 23.5 pM (Bmax = 36.4 fmol/mg of protein) and to 86 +/- 3% of the sites with a Kd of 1.9 nM (Bmax = 220.1 fmol/mg of protein). The fragments NPY 13-36 and PYY 13-36 were able to compete with 10 pM Tyr1-125I-NPY for essentially all the binding sites. The fragments were 1 to 2 orders of magnitude less potent than the native peptides. Approximately 50% of the moderate affinity sites, but not the high affinity sites, were reversibly lost in the presence of 5'-guanylyl imidophosphate (Gpp(NH)p), a nonhydrolyzable analog of GTP. Kinetic studies revealed that Tyr1-125I-NPY dissociation could be best described by three dissociation rates. The proportions of slow and intermediate dissociation matched the proportions of moderate and high affinity binding sites, respectively, as suggested by equilibrium studies. There also existed a phase of fast dissociation. When Gpp(NH)p was added during dissociation, the proportion of slow dissociation decreased to the same extent that the fast dissociation was increased. However, the proportion of intermediate dissociation did not change. We propose that rat brain contains a minor population of high affinity NPY binding sites with an intermediate dissociation rate and no sensitivity to Gpp(NH)p.

  1. Determination of multiple-clone infection at allelic dimorphism site of Plasmodium vivax merozoite surface protein-1 in the Republic of Korea by pyrosequencing assay.

    PubMed

    Dinzouna-Boutamba, Sylvatrie-Danne; Lee, Sanghyun; Son, Ui-Han; Yun, Hae Soo; Joo, So-Young; Jeong, Sookwan; Rhee, Man Hee; Kwak, Dongmi; Xuan, Xuenan; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung

    2017-08-25

    Allelic diversity leading to multiple gene polymorphisms of vivax malaria parasites has been shown to greatly contribute to antigenic variation and drug resistance, increasing the potential for multiple-clone infections within the host. Therefore, to identify multiple-clone infections and the predominant haplotype of Plasmodium vivax in a South Korean population, P. vivax merozoite surface protein-1 (PvMSP-1) was analyzed by pyrosequencing. Pyrosequencing of 156 vivax malaria-infected samples yielded 97 (62.18%) output pyrograms showing two main types of peak patterns of the dimorphic allele for threonine and alanine (T1476A). Most of the samples evaluated (88.66%) carried multiple-clone infections (wild- and mutant-types), whereas 11.34% of the same population carried only the mutant-type (1476A). In addition, each allele showed a high frequency of guanine (G) base substitution at both the first and third positions (86.07% and 81.13%, respectively) of the nucleotide combinations. Pyrosequencing of the PvMSP-1 42-kDa fragment revealed a heterogeneous parasite population, with the mutant-type dominant compared to the wild-type. Understanding the genetic diversity and multiple-clone infection rates may lead to improvements in vivax malaria prevention and strategic control plans. Further studies are needed to improve the efficacy of the pyrosequencing assay with large sample sizes and additional nucleotide positions. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

    PubMed Central

    Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

    1992-01-01

    Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

  3. 8-Chloro-adenosine-induced E2F1 promotes p14ARF gene activation in H1299 cells through displacing Sp1 from multiple overlapping E2F1/Sp1 sites.

    PubMed

    Zhang, Hai-Jun; Li, Wen-Juan; Yang, Sheng-Yong; Li, Shu-Yan; Ni, Ju-Hua; Jia, Hong-Ti

    2009-02-15

    The regulation of p14ARF gene by E2F transcription factor, which differs from that of classical E2F targets, has recently been attributed to a variant E2F-response element. However, promoter assays suggest multiple elements present in the p14ARF promoter and argue against the idea that the ARF promoter has a unique ability to distinguish between aberrant and physiological levels of E2F1. Therefore, the functional characterization of the promoter still needs to be done. We demonstrate that at least two overlapping E2F1/Sp1 binding sites are present in the p14ARF promoter, and E2F1 activates the promoter through displacing constitutive Sp1 from the overlapping sites. We found that 8-chloro-adenosine (a metabolite of 8-Cl-cAMP) exposure induced the p14ARF gene in human lung cancer H1299 cells, followed by increased expression of E2F1 and constitutive expression of Sp1. The combination of cotransfection and electrophoretic mobility shift assay (EMSA) indicated that constitutive binding of Sp1 to the overlapping sites contributed to a constitutive expression of the ARF gene in unexposed H1299, whereas displacing Sp1 from the overlapping sites by E2F1 promoted the gene activation after exposure. EMSA and chromatin immunoprecipitation revealed increased association of E2F1 with the overlapping sites in the active promoter in 8-Cl-Ado-exposed cells. Together, these data suggest that the overlapping E2F1/Sp1 site, being present in multiple copies in the p14ARF promoter, may serve as the targets for both E2F1 and Sp1, thereby playing a crucial role in response to some oncogenic signals and stimulators, which activate the ARF gene through inducing E2F in the cell.

  4. Identification of a multifunctional docking site on the catalytic unit of phosphodiesterase-4 (PDE4) that is utilised by multiple interaction partners

    PubMed Central

    Houslay, Kirsty F.; Christian, Frank; MacLeod, Ruth; Adams, David R.; Houslay, Miles D.

    2017-01-01

    Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold β-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif. PMID:27993970

  5. Ropizine concurrently enhances and inhibits ( sup 3 H) dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

    SciTech Connect

    Canoll, P.D.; Smith, P.R.; and Musacchio, J.M. )

    1990-01-01

    Ropizine produces a simultaneous enhancement and inhibition of ({sup 3}H) dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity ({sup 3}H)DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances ({sup 3}H)DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+){minus} pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of ({sup 3}H)DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.

  6. Validation of a radiosonde-based cloud layer detection method against a ground-based remote sensing method at multiple ARM sites

    NASA Astrophysics Data System (ADS)

    Zhang, Jinqiang; Li, Zhanqing; Chen, Hongbin; Cribb, Maureen

    2013-01-01

    Cloud vertical structure is a key quantity in meteorological and climate studies, but it is also among the most difficult quantities to observe. In this study, we develop a long-term (10 years) radiosonde-based cloud profile product for the U.S. Department of Energy's Atmospheric Radiation Measurement (ARM) program Southern Great Plains (SGP), Tropical Western Pacific (TWP), and North Slope of Alaska (NSA) sites and a shorter-term product for the ARM Mobile Facility (AMF) deployed in Shouxian, Anhui Province, China (AMF-China). The AMF-China site was in operation from 14 May to 28 December 2008; the ARM sites have been collecting data for over 15 years. The Active Remote Sensing of Cloud (ARSCL) value-added product (VAP), which combines data from the 95-GHz W-band ARM Cloud Radar (WACR) and/or the 35-GHz Millimeter Microwave Cloud Radar (MMCR), is used in this study to validate the radiosonde-based cloud layer retrieval method. The performance of the radiosonde-based cloud layer retrieval method applied to data from different climate regimes is evaluated. Overall, cloud layers derived from the ARSCL VAP and radiosonde data agree very well at the SGP and AMF-China sites. At the TWP and NSA sites, the radiosonde tends to detect more cloud layers in the upper troposphere.

  7. Two-phase positive inotropic effects of ouabain and the presence of multiple classes of ouabain binding sites in the ferret heart

    SciTech Connect

    Ng, Y.C.; Akera, T.

    1986-03-05

    Characteristics of more than one class of ouabain receptors which appear to exist in ferret heart were examined. In isolated papillary muscle, 1 to 30 nM ouabain produced a positive inotropic effect in the presence of 5 ..mu..M propranolol and 2 ..mu..M phentolamine. Higher concentrations of ouabain (0.1 to 10 ..mu..M) produced an additional and prominent inotropic effect. In partially purified Na, K-ATPase, ouabain caused a monophasic inhibition; however, the concentration-inhibition curve spanned over 5 log units, indicating that ouabain is interacting with more than a single class of the enzyme. Scatchard analysis of specific /sup 3/H-ouabain binding revealed approximately equal abundance of high and low affinity binding sites. The K/sub D/ value for high affinity sites was approximately 20 nM whereas that for low affinity sites was about 45 times higher. When phosphoenzyme was formed in the presence of (..gamma..-/sup 32/P)-ATP, Mg/sup 2 +/ and Na/sup +/ and subjected to SDS gel electrophoresis, two distinct K/sup +/-sensitive bands with about 100,000 dalton molecular weight were detected. Molecular weight difference between these two bands was approximately 2500 dalton. Phosphorylation of either band was abolished by 1 ..mu..M