Sample records for target sites including

  1. Centredale Manor Superfund Site in Rhode Island included on EPA List of Targeted for Immediate Attention

    EPA Pesticide Factsheets

    Today, the U.S. Environmental Protection Agency released the list of Superfund sites that Administrator Pruitt has targeted for immediate and intense attention. The Centredale Manor Restoration Project superfund site is one of the 21 sites on the list.

  2. Mohawk Tannery Hazardous Waste Site in New Hampshire included on EPA List of Targeted for Immediate Attention

    EPA Pesticide Factsheets

    Today, the U.S. Environmental Protection Agency released the list of Superfund sites that Administrator Pruitt has targeted for immediate and intense attention. The former Mohawk Tannery facility (a.k.a. Granite State Leathers) is one of the 21 sites on th

  3. SeedVicious: Analysis of microRNA target and near-target sites.

    PubMed

    Marco, Antonio

    2018-01-01

    Here I describe seedVicious, a versatile microRNA target site prediction software that can be easily fitted into annotation pipelines and run over custom datasets. SeedVicious finds microRNA canonical sites plus other, less efficient, target sites. Among other novel features, seedVicious can compute evolutionary gains/losses of target sites using maximum parsimony, and also detect near-target sites, which have one nucleotide different from a canonical site. Near-target sites are important to study population variation in microRNA regulation. Some analyses suggest that near-target sites may also be functional sites, although there is no conclusive evidence for that, and they may actually be target alleles segregating in a population. SeedVicious does not aim to outperform but to complement existing microRNA prediction tools. For instance, the precision of TargetScan is almost doubled (from 11% to ~20%) when we filter predictions by the distance between target sites using this program. Interestingly, two adjacent canonical target sites are more likely to be present in bona fide target transcripts than pairs of target sites at slightly longer distances. The software is written in Perl and runs on 64-bit Unix computers (Linux and MacOS X). Users with no computing experience can also run the program in a dedicated web-server by uploading custom data, or browse pre-computed predictions. SeedVicious and its associated web-server and database (SeedBank) are distributed under the GPL/GNU license.

  4. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

    PubMed Central

    Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

    2015-01-01

    Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations. PMID:25985872

  5. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  6. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-01-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  7. Photoaffinity labeling in target- and binding-site identification

    PubMed Central

    Smith, Ewan; Collins, Ian

    2015-01-01

    Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

  8. Transcription factor target site search and gene regulation in a background of unspecific binding sites.

    PubMed

    Hettich, J; Gebhardt, J C M

    2018-06-02

    Response time and transcription level are vital parameters of gene regulation. They depend on how fast transcription factors (TFs) find and how efficient they occupy their specific target sites. It is well known that target site search is accelerated by TF binding to and sliding along unspecific DNA and that unspecific associations alter the occupation frequency of a gene. However, whether target site search time and occupation frequency can be optimized simultaneously is mostly unclear. We developed a transparent and intuitively accessible state-based formalism to calculate search times to target sites on and occupation frequencies of promoters of arbitrary state structure. Our formalism is based on dissociation rate constants experimentally accessible in live cell experiments. To demonstrate our approach, we consider promoters activated by a single TF, by two coactivators or in the presence of a competitive inhibitor. We find that target site search time and promoter occupancy differentially vary with the unspecific dissociation rate constant. Both parameters can be harmonized by adjusting the specific dissociation rate constant of the TF. However, while measured DNA residence times of various eukaryotic TFs correspond to a fast search time, the occupation frequencies of target sites are generally low. Cells might tolerate low target site occupancies as they enable timely gene regulation in response to a changing environment. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  9. Thermodynamics of DNA target site recognition by homing endonucleases

    PubMed Central

    Eastberg, Jennifer H.; Smith, Audrey McConnell; Zhao, Lei; Ashworth, Justin; Shen, Betty W.; Stoddard, Barry L.

    2007-01-01

    The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of ΔH and TΔS are not strongly correlated with the overall extent of DNA bending, unfavorable ΔHbinding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific ΔSbinding. PMID:17947319

  10. Renton's Quendall Terminals on List of EPA Superfund Sites Targeted for Immediate, Intense Attention

    EPA Pesticide Factsheets

    EPA released the list of Superfund sites that Administrator Pruitt has targeted for intense and immediate attention, including the Quendall Terminals Site, a former creosote facility on the shore of Lake Washington in Renton, Washington.

  11. Prostate Cancer Clinical Consortium Clinical Research Site:Targeted Therapies

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-2-0159 TITLE: Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies PRINCIPAL INVESTIGATOR...Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5b. GRANT NUMBER... therapy resistance/sensitivity, identification of new therapeutic targets through high quality genomic analyses, providing access to the highest quality

  12. TarPmiR: a new approach for microRNA target site prediction.

    PubMed

    Ding, Jun; Li, Xiaoman; Hu, Haiyan

    2016-09-15

    The identification of microRNA (miRNA) target sites is fundamentally important for studying gene regulation. There are dozens of computational methods available for miRNA target site prediction. Despite their existence, we still cannot reliably identify miRNA target sites, partially due to our limited understanding of the characteristics of miRNA target sites. The recently published CLASH (crosslinking ligation and sequencing of hybrids) data provide an unprecedented opportunity to study the characteristics of miRNA target sites and improve miRNA target site prediction methods. Applying four different machine learning approaches to the CLASH data, we identified seven new features of miRNA target sites. Combining these new features with those commonly used by existing miRNA target prediction algorithms, we developed an approach called TarPmiR for miRNA target site prediction. Testing on two human and one mouse non-CLASH datasets, we showed that TarPmiR predicted more than 74.2% of true miRNA target sites in each dataset. Compared with three existing approaches, we demonstrated that TarPmiR is superior to these existing approaches in terms of better recall and better precision. The TarPmiR software is freely available at http://hulab.ucf.edu/research/projects/miRNA/TarPmiR/ CONTACTS: haihu@cs.ucf.edu or xiaoman@mail.ucf.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  13. 76 FR 35026 - Hutchinson Technology, Inc., Including On-Site Workers Leased From Doherty, Including Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-15

    ... Technology, Inc., Including On-Site Workers Leased From Doherty, Including Workers Whose Unemployment... Technology, Inc., Including On-Site Workers Leased From Doherty, Including Workers Whose Unemployment..., Minnesota locations of the subject firm had their wages reported under a separate unemployment insurance (UI...

  14. Retention of ferrofluid aggregates at the target site during magnetic drug targeting

    NASA Astrophysics Data System (ADS)

    Asfer, Mohammed; Saroj, Sunil Kumar; Panigrahi, Pradipta Kumar

    2017-08-01

    The present study reports the retention dynamics of a ferrofluid aggregate localized at the target site inside a glass capillary (500 × 500 μm2 square cross section) against a bulk flow of DI water (Re = 0.16 and 0.016) during the process of magnetic drug targeting (MDT). The dispersion dynamics of iron oxide nanoparticles (IONPs) into bulk flow for different initial size of aggregate at the target site is reported using the brightfield visualization technique. The flow field around the aggregate during the retention is evaluated using the μPIV technique. IONPs at the outer boundary experience a higher shear force as compared to the magnetic force, resulting in dispersion of IONPs into the bulk flow downstream to the aggregate. The blockage effect and the roughness of the outer boundary of the aggregate resulting from chain like clustering of IONPs contribute to the flow recirculation at the downstream region of the aggregate. The entrapment of seeding particles inside the chain like clusters of IONPs at the outer boundary of the aggregate reduces the degree of roughness resulting in a streamlined aggregate at the target site at later time. The effect of blockage, structure of the aggregate, and disturbed flow such as recirculation around the aggregate are the primary factors, which must be investigated for the effectiveness of the MDT process for in vivo applications.

  15. Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies

    DTIC Science & Technology

    2017-10-01

    AWARD NUMBER: W81XWH-14-2-0159 TITLE: Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies PRINCIPAL INVESTIGATOR...Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for...AND SUBTITLE Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT

  16. Target-mediated drug disposition model for drugs with two binding sites that bind to a target with one binding site.

    PubMed

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2017-10-01

    The paper extended the TMDD model to drugs with two identical binding sites (2-1 TMDD). The quasi-steady-state (2-1 QSS), quasi-equilibrium (2-1 QE), irreversible binding (2-1 IB), and Michaelis-Menten (2-1 MM) approximations of the model were derived. Using simulations, the 2-1 QSS approximation was compared with the full 2-1 TMDD model. As expected and similarly to the standard TMDD for monoclonal antibodies (mAb), 2-1 QSS predictions were nearly identical to 2-1 TMDD predictions, except for times of fast changes following initiation of dosing, when equilibrium has not yet been reached. To illustrate properties of new equations and approximations, several variations of population PK data for mAbs with soluble (slow elimination of the complex) or membrane-bound (fast elimination of the complex) targets were simulated from a full 2-1 TMDD model and fitted to 2-1 TMDD models, to its approximations, and to the standard (1-1) QSS model. For a mAb with a soluble target, it was demonstrated that the 2-1 QSS model provided nearly identical description of the observed (simulated) free drug and total target concentrations, although there was some minor bias in predictions of unobserved free target concentrations. The standard QSS approximation also provided a good description of the observed data, but was not able to distinguish between free drug concentrations (with no target attached and both binding site free) and partially bound drug concentrations (with one of the binding sites occupied by the target). For a mAb with a membrane-bound target, the 2-1 MM approximation adequately described the data. The 2-1 QSS approximation converged 10 times faster than the full 2-1 TMDD, and its run time was comparable with the standard QSS model.

  17. A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

    PubMed

    Menchon, Grégory; Prota, Andrea E; Lucena-Agell, Daniel; Bucher, Pascal; Jansen, Rolf; Irschik, Herbert; Müller, Rolf; Paterson, Ian; Díaz, J Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

    2018-05-29

    Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

  18. Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

    PubMed Central

    Herbert, Martha

    2017-01-01

    Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH) receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw) and modified (cooked and roasted) foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease. PMID:28894619

  19. RNase L targets distinct sites in influenza A virus RNAs.

    PubMed

    Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

    2015-03-01

    Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Engineering synthetic TAL effectors with orthogonal target sites

    PubMed Central

    Garg, Abhishek; Lohmueller, Jason J.; Silver, Pamela A.; Armel, Thomas Z.

    2012-01-01

    The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression. PMID:22581776

  1. Influence of quasi-specific sites on kinetics of target DNA search by a sequence-specific DNA-binding protein.

    PubMed

    Kemme, Catherine A; Esadze, Alexandre; Iwahara, Junji

    2015-11-10

    Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such "quasi-specific" sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1's association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

  2. Influence of Quasi-Specific Sites on Kinetics of Target DNA Search by a Sequence-Specific DNA-Binding Protein

    PubMed Central

    2015-01-01

    Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such “quasi-specific” sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1’s association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins. PMID:26502071

  3. Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

    PubMed

    Rockah-Shmuel, Liat; Tawfik, Dan S

    2012-12-01

    DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

  4. Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

    PubMed Central

    Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

    2013-01-01

    Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

  5. Characteristics of Food Industry Web Sites and "Advergames" Targeting Children

    ERIC Educational Resources Information Center

    Culp, Jennifer; Bell, Robert A.; Cassady, Diana

    2010-01-01

    Objective: To assess the content of food industry Web sites targeting children by describing strategies used to prolong their visits and foster brand loyalty; and to document health-promoting messages on these Web sites. Design: A content analysis was conducted of Web sites advertised on 2 children's networks, Cartoon Network and Nickelodeon. A…

  6. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

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

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein ismore » designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.« less

  7. Initiatives to Develop Web Sites Including Information about Brownfields Properties

    EPA Pesticide Factsheets

    This web site was created to assist in planning, designing, and operating web sites that include information about individual brownfields properties. The report is of value to parties designing or managing such sites.

  8. Spy: a new group of eukaryotic DNA transposons without target site duplications.

    PubMed

    Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

    2014-06-24

    Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites.

    PubMed

    Betel, Doron; Koppal, Anjali; Agius, Phaedra; Sander, Chris; Leslie, Christina

    2010-01-01

    mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.

  10. Target Site Recognition by a Diversity-Generating Retroelement

    PubMed Central

    Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

    2011-01-01

    Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

  11. Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

    PubMed Central

    Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M.; Jenny, Patrick; Hardt, Wolf-Dietrich

    2012-01-01

    Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. PMID:22911370

  12. Initial basalt target site selection evaluation for the Mars penetrator drop test

    NASA Technical Reports Server (NTRS)

    Bunch, T. E.; Quaide, W. L.; Polkowski, G.

    1976-01-01

    Potential basalt target sites for an air drop penetrator test were described and the criteria involved in site selection were discussed. A summary of the background field geology and recommendations for optimum sites are also presented.

  13. Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

    PubMed

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2018-02-01

    The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

  14. 75 FR 66795 - TTM Technologies, Including On-Site Leased Workers From Kelly Services, Aerotek, and an On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-29

    ... follows: ``All workers TTM Technologies, including on-site leased workers from Kelly Services and Aerotek... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-64,993] TTM Technologies, Including On-Site Leased Workers From Kelly Services, Aerotek, and an On-Site Leased Worker From Orbotech...

  15. The impact of target site accessibility on the design of effective siRNAs.

    PubMed

    Tafer, Hakim; Ameres, Stefan L; Obernosterer, Gregor; Gebeshuber, Christoph A; Schroeder, Renée; Martinez, Javier; Hofacker, Ivo L

    2008-05-01

    Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.

  16. Recovery of perennial vegetation in military target sites in the eastern Mohave Desert, Arizona

    USGS Publications Warehouse

    Steiger, John W.; Webb, Robert H.

    2000-01-01

    The effect of the age of geomorphic surfaces on the recovery of desert vegetation in military target sites was studied in the Mohave and Cerbat Mountains of northwestern Arizona. The target sites were cleared of all vegetation during military exercises in 1942-1943 and have not been subsequently disturbed. The degree of recovery was measured by calculating percentage-similarity (PS) and correlation-coefficient indices on the basis of differences in cover, density, and volume of species growing in and out of each target site. PS values, ranging from 22.7 to 95.1 percent (100 percent = identical composition), indicate a wide range of recovery that is partially controlled by the edaphic properties of the geomorphic surfaces. Statistical analyses show a strong pattern that indicates a greater variability in the degree of recovery for sites on older surfaces than on younger surfaces and a weak pattern that indicates an inverse relation between the degree of recovery and geomorphic age. Comparisons of the different effects of target site construction on the edaphic characteristics of each target site provides an explanation for these patterns and suggests the soil properties critical to the recovery process. Statistically significant negative or positive response to disturbance for most species are independent of the age of the geomorphic surfaces; however, there is strong evidence for a shift in response for the common perennial species Acamptopappus sphaerocephalus, and to a lesser extent, Salazaria mexicana, Encelia farinosa, and Coldenia canescens, among different geomorphic surfaces.

  17. 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.

  18. Characteristics of food industry web sites and "advergames" targeting children.

    PubMed

    Culp, Jennifer; Bell, Robert A; Cassady, Diana

    2010-01-01

    To assess the content of food industry Web sites targeting children by describing strategies used to prolong their visits and foster brand loyalty; and to document health-promoting messages on these Web sites. A content analysis was conducted of Web sites advertised on 2 children's networks, Cartoon Network and Nickelodeon. A total of 290 Web pages and 247 unique games on 19 Internet sites were examined. Games, found on 81% of Web sites, were the most predominant promotion strategy used. All games had at least 1 brand identifier, with logos being most frequently used. On average Web sites contained 1 "healthful" message for every 45 exposures to brand identifiers. Food companies use Web sites to extend their television advertising to promote brand loyalty among children. These sites almost exclusively promoted food items high in sugar and fat. Health professionals need to monitor food industry marketing practices used in "new media." Published by Elsevier Inc.

  19. Widespread occurrence of both metabolic and target-site herbicide resistance mechanisms in Lolium rigidum populations.

    PubMed

    Han, Heping; Yu, Qin; Owen, Mechelle J; Cawthray, Gregory R; Powles, Stephen B

    2016-02-01

    Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations. The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations. This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts. © 2015 Society of Chemical Industry.

  20. 77 FR 9969 - Clow Water Systems Company Including On-Site Leased Workers From Carol Harris Stafffing Including...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... Company Including On-Site Leased Workers From Carol Harris Stafffing Including Workers Whose Unemployment... unemployment insurance (UI) tax account under the name McWane, Inc. Accordingly, the Department is amending this certification to include workers of the subject firm whose unemployment insurance (UI) wages are...

  1. Repopulation of calibrations with samples from the target site: effect of the size of the calibration.

    NASA Astrophysics Data System (ADS)

    Guerrero, C.; Zornoza, R.; Gómez, I.; Mataix-Solera, J.; Navarro-Pedreño, J.; Mataix-Beneyto, J.; García-Orenes, F.

    2009-04-01

    squares regression, and leave-one-out cross validation as methods of calibration. Two methods were used to select the different quantities (size of models) of samples: (1) Based on Characteristics of Spectra (BCS), and (2) Based on NKj Values of Samples (BVS). Both methods tried to select representative samples. Each of the calibrations (containing the 5, 10, 25, 50, 75 or 100% of the total samples of the library) was repopulated with samples from the target site and then recalibrated (by leave-one-out cross validation). This procedure was sequential. In each step, 2 samples from the target site were added to the models, and then recalibrated. This process was repeated successively 10 times, being 20 the total number of samples added. A local model was also created with the 20 samples used for repopulation. The repopulated, non-repopulated and local calibrations were used to predict the NKj content in those samples from the target site not included in repopulations. For the measurement of the accuracy of the predictions, the r2, RMSEP and slopes were calculated comparing predicted with analysed NKj values. This scheme was repeated for each of the four target sites studied. In general, scarce differences can be found between results obtained with BCS and BVS models. We observed that the repopulation of models increased the r2 of the predictions in sites 1 and 3. The repopulation caused scarce changes of the r2 of the predictions in sites 2 and 4, maybe due to the high initial values (using non-repopulated models r2 >0.90). As consequence of repopulation, the RMSEP decreased in all the sites except in site 2, where a very low RMESP was obtained before the repopulation (0.4 g×kg-1). The slopes trended to approximate to 1, but this value was reached only in site 4 and after the repopulation with 20 samples. In sites 3 and 4, accurate predictions were obtained using the local models. Predictions obtained with models using similar size of samples (similar %) were averaged with

  2. Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random.

    PubMed Central

    Furano, A V; Somerville, C C; Tsichlis, P N; D'Ambrosio, E

    1986-01-01

    The long interspersed repeated DNA family of rats (LINE or L1Rn family) contains about 40,000 6.7-kilobase (kb) long members (1). LINE members may be currently mobile since their presence or absence causes allelic variation at three single copy loci (2, 3): insulin 1, Moloney leukemia virus integration 2 (Mlvi-2) (4), and immunoglobulin heavy chain (Igh). To characterize target sites for LINE insertion, we compared the DNA sequences of the unoccupied Mlvi-2 target site, its LINE-containing allele, and several other LINE-containing sites. Although not homologous overall, the target sites share three characteristics: First, depending on the site, they are from 68% to 86% (A+T) compared to 58% (A+T) for total rat DNA (5). Depending on the site, a 7- to 15-bp target site sequence becomes duplicated and flanks the inserted LINE member. The second is a version (0 or 1 mismatch) of the hexanucleotide, TACTCA, which is also present in the LINE member, in a highly conserved region located just before the A-rich right end of the LINE member. The third is a stretch of alternating purine/pyrimidine (PQ). The A-rich right ends of different LINE members vary in length and composition, and the sequence of a particularly long one suggests that it contains the A-rich target site from a previous transposition. PMID:3012480

  3. Barriers to Liposomal Gene Delivery: from Application Site to the Target.

    PubMed

    Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

    2016-01-01

    Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

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

    PubMed

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

    2015-12-08

    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.

  5. 75 FR 16513 - B&C Corporation, JR Engineering Division, Including B&C Distribution Center, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-01

    ... Engineering Division, Including B&C Distribution Center, Including On-Site Leased Workers From B&C Services, Inc., Barberton, OH; Amended Certification Regarding Eligibility To Apply for Worker Adjustment... Department of Labor issued a Certification of Eligibility to Apply for Worker Adjustment Assistance on...

  6. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  7. 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

  8. mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation.

    PubMed

    Levy, Oren; Zhao, Weian; Mortensen, Luke J; Leblanc, Sarah; Tsang, Kyle; Fu, Moyu; Phillips, Joseph A; Sagar, Vinay; Anandakumaran, Priya; Ngai, Jessica; Cui, Cheryl H; Eimon, Peter; Angel, Matthew; Lin, Charles P; Yanik, Mehmet Fatih; Karp, Jeffrey M

    2013-10-03

    Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

  9. Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

    PubMed Central

    Pierce, M L; Ruffner, D E

    1998-01-01

    Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

  10. 75 FR 879 - National Semiconductor Corporation Arlington Manufacturing Site Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-06

    ... Foods, Allied Barton Security, ASIL, ASML and Construction Mechanical Systems Arlington, TX; Amended... leased workers from ASML and Construction Mechanical Systems were employed on-site at the Arlington... Department is amending the certification to include workers leased from ASML and Construction Mechanical...

  11. LuciPHOr: Algorithm for Phosphorylation Site Localization with False Localization Rate Estimation Using Modified Target-Decoy Approach*

    PubMed Central

    Fermin, Damian; Walmsley, Scott J.; Gingras, Anne-Claude; Choi, Hyungwon; Nesvizhskii, Alexey I.

    2013-01-01

    The localization of phosphorylation sites in peptide sequences is a challenging problem in large-scale phosphoproteomics analysis. The intense neutral loss peaks and the coexistence of multiple serine/threonine and/or tyrosine residues are limiting factors for objectively scoring site patterns across thousands of peptides. Various computational approaches for phosphorylation site localization have been proposed, including Ascore, Mascot Delta score, and ProteinProspector, yet few address direct estimation of the false localization rate (FLR) in each experiment. Here we propose LuciPHOr, a modified target-decoy-based approach that uses mass accuracy and peak intensities for site localization scoring and FLR estimation. Accurate estimation of the FLR is a difficult task at the individual-site level because the degree of uncertainty in localization varies significantly across different peptides. LuciPHOr carries out simultaneous localization on all candidate sites in each peptide and estimates the FLR based on the target-decoy framework, where decoy phosphopeptides generated by placing artificial phosphorylation(s) on non-candidate residues compete with the non-decoy phosphopeptides. LuciPHOr also reports approximate site-level confidence scores for all candidate sites as a means to localize additional sites from multiphosphorylated peptides in which localization can be partially achieved. Unlike the existing tools, LuciPHOr is compatible with any search engine output processed through the Trans-Proteomic Pipeline. We evaluated the performance of LuciPHOr in terms of the sensitivity and accuracy of FLR estimates using two synthetic phosphopeptide libraries and a phosphoproteomic dataset generated from complex mouse brain samples. PMID:23918812

  12. An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

    PubMed

    Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

    2017-04-01

    Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

    PubMed Central

    Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.

    2009-01-01

    The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits. PMID:19321668

  14. E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

    PubMed

    Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

    2017-01-01

    Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

  15. Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides.

    PubMed

    Pang, Yuan-Ping; Brimijoin, Stephen; Ragsdale, David W; Zhu, Kun Yan; Suranyi, Robert

    2012-04-01

    Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market.

  16. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

    PubMed

    Lee, Ciaran M; Davis, Timothy H; Bao, Gang

    2018-04-01

    What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

  17. Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

    PubMed Central

    Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

    2013-01-01

    Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

  18. 76 FR 5833 - Polaris Industries, Including On-Site Leased Workers From Westaff, Supply Technologies, Aerotek...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-02

    ..., Including On-Site Leased Workers From Westaff, Supply Technologies, Aerotek, and Securitas Security Services..., including on-site leased workers from Westaff, Supply Technologies, Aerotek and Securitas Security Services... was amended on December 6, 2010 to include on- site leased workers from Supply Technologies. The...

  19. Novel and Viable Acetylcholinesterase Target Site for Developing Effective and Environmentally Safe Insecticides

    PubMed Central

    Pang, Yuan-Ping; Brimijoin, Stephen; Ragsdale, David W; Zhu, Kun Yan; Suranyi, Robert

    2012-01-01

    Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market. PMID:22280344

  20. Rational modification of protein stability by targeting surface sites leads to complicated results

    PubMed Central

    Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

    2013-01-01

    The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

  1. Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion.

    PubMed

    Gladyshev, Eugene A; Arkhipova, Irina R

    2009-12-15

    Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: site-specific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.

  2. 75 FR 32221 - Whirlpool Corporation, Evansville Division, Including On-Site Leased Workers from Andrews...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-07

    ..., Evansville Division, Including On-Site Leased Workers from Andrews International, Inc., Evansville, IN... that workers leased from Andrews International, Inc. were employed on-site at the Evansville, Indiana... findings, the Department is amending this certification to include leased workers from Andrews...

  3. 78 FR 1252 - CalAmp Wireless Networks Corporation (CWNC), Satellite Products Division, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-08

    ... Select Staffing, Oxnard, CA; CalAmp Wireless Networks Corporation (CWNC), Including On- Site Leased... Division, including on-site leased workers from Select Staffing, Oxnard, California (TA-W-80,399). The...-site leased workers from Select Staffing, Oxnard, California (TA-W-80,399) and CalAmp Wireless Networks...

  4. A mathematical model of single target site location by Brownian movement in subcellular compartments.

    PubMed

    Kuthan, Hartmut

    2003-03-07

    The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near

  5. Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

    PubMed

    Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

    2017-06-22

    Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI

    PubMed Central

    Shen, Betty; Heiter, Daniel F.; Chan, Siu-Hong; Wang, Hua; Xu, Shuang-Yong; Morgan, Richard D.; Wilson, Geoffrey G.; Stoddard, Barry L.

    2010-01-01

    The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight base pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 Å resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a ββα-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand-cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA basepairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in non-canonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures. PMID:20541511

  7. 77 FR 53914 - Horton Automatics, Inc., a Subsidiary of Overhead Door Corporation Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-04

    ...., a Subsidiary of Overhead Door Corporation Including On-Site Leased Workers From Remedy Intelligent..., including on-site leased workers from Remedy Intelligent Staffing, Corpus Christi, Texas. The workers are... Automatics, Inc., a subsidiary of Overhead Door Corporation, including on-site leased workers from Remedy...

  8. 76 FR 19466 - Masco Builder Cabinet Group Including On-Site Leased Workers From Reserves Network, Reliable...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-07

    ... Builder Cabinet Group Including On-Site Leased Workers From Reserves Network, Reliable Staffing, and Third Dimension Waverly, OH; Masco Builder Cabinet Group Including On-Site Leased Workers From Reserves Network... Group including on-site leased workers from Reserves Network, Jackson, Ohio. The workers produce...

  9. 78 FR 49292 - Northshore Mining Company, a Subsidiary of Cliffs Natural Resources, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-13

    ... Mining Company, a Subsidiary of Cliffs Natural Resources, Including On-Site Leased Workers From Vanhouse... Cliffs Natural Resources, Including On- Site Leased Workers From Vanhouse, Express Employment and Our... Natural Resources, including on-site leased workers from VanHouse and Express Employment, Silver Bay...

  10. 78 FR 19530 - Eastman Kodak Company (GCG), Electrographic Print Solutions, Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-01

    ... Kodak Company (GCG), Electrographic Print Solutions, Including On-Site Leased Workers From Adecco and Datrose, Spencerport, New York; Eastman Kodak Company, IPS, Including On-Site Leased Workers From Adecco..., 2011, applicable to workers of Eastman Kodak Company (GCG), Electrographic Print Solutions, including...

  11. Analysis on establishing Chang'E-3 landing site as a reflectance calibration target

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Fu, Xiaohui; Zeng, Xingguo; Yao, Meijuan; Zhang, Hongbo; Su, Yan; Zhao, Shu; Xue, Xiping; Li, Chunlai; Zou, Yongliao

    2015-04-01

    Recent lunar orbital observations suggested that the surface reflectance calculated based on the Apollo 16 standard area and Apollo 16 sample laboratory measurement is significantly different from its true value [1-3], one reason is the composition and maturity differences between the 62231 sampling site and the Apollo 16 standard site existed, the other reason is the physical properties of the returned lunar sample, such as porosity, have been changed during the sampling operations. So more new standard targets on the Moon, besides the widely used Apollo 16 area, are needed for imaging spectrometers on lunar missions to improve their reflectance calibration accuracies. The Chang'E-3 VIS/NIR Imaging Spectrometer (VNIS), which is just fixed at the front of the Yutu rover [4], equipped with a white spectralon panel as reflectance calibration standard, can perform in situ multispectral observations around the Chang'E-3 landing site without altering the physical and mineralogical natures of lunar soils. Therefore, it provides an opportunity to establish a new reliable standard target for in-flight reflectance calibration. The reflectance calibration target should be compositional homogeneous, the topography of which must be flat, and the reflectance should be identical with no nearby units of other different materials. As we have known, Chang'e-3 probe landed on the Mare Imbrium basin in the east part of Sinus Iridum, the landing site is relatively flat at a spatial coverage of ~660km2, and this region belongs to Eratosthenian low-Ti/high-Ti mare basalts [5-6]. According to much higher resolution topography data, elemental data and reflectance data of Chang'E-2 and Chang'E-3[7-8], we preliminary analyse the possibility on establishing Chang'E-3 landing site as a reflectance calibration target. Firstly, the overall terrain of the 4 km×4 km area around the landing site is flat, but there are still three bigger craters existed. Secondly, the composition on Chang'E-3

  12. Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

    PubMed Central

    Mondal, Anupam; Bhattacherjee, Arnab

    2015-01-01

    DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

  13. 78 FR 8586 - PEPSICO, Inc., Business & Information Solutions (BIS) Division Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-06

    ...., Business & Information Solutions (BIS) Division, including on-site leased workers of Cognizant Technology..., Inc., Business & Information Solutions (BIS) Division Including On-Site Leased Workers From Procurestaff, Cognizant, Infosys, Wipro, and TCS; Plano, TX; PEPSICO, Inc., Business & Information Solutions...

  14. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

    PubMed Central

    Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

    2015-01-01

    Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

  15. Cryostat including heater to heat a target

    DOEpatents

    Pehl, Richard H.; Madden, Norman W.; Malone, Donald F.

    1990-01-01

    A cryostat is provided which comprises a vacuum vessel; a target disposed within the vacuum vessel; a heat sink disposed within the vacuum vesssel for absorbing heat from the detector; a cooling mechanism for cooling the heat sink; a cryoabsorption mechanism for cryoabsorbing residual gas within the vacuum vessel; and a heater for maintaining the target above a temperature at which the residual gas is cryoabsorbed in the course of cryoabsorption of the residual gas by the cryoabsorption mechanism.

  16. 78 FR 12358 - UBS Financial Services, Inc., Wealth Management Americas Operations, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-22

    ..., Inc., Wealth Management Americas Operations, Including On-Site Leased Workers From Leafstone... Services, Inc., Wealth Management Americas Operations (UBS), Weehawken, New Jersey. The workers are engaged... to include all leased workers on-site at UBS Financial Services, Inc., Wealth Management Americas...

  17. Vegetation and the importance of insecticide-treated target siting for control of Glossina fuscipes fuscipes.

    PubMed

    Esterhuizen, Johan; Njiru, Basilio; Vale, Glyn A; Lehane, Michael J; Torr, Stephen J

    2011-09-01

    Control of tsetse flies using insecticide-treated targets is often hampered by vegetation re-growth and encroachment which obscures a target and renders it less effective. Potentially this is of particular concern for the newly developed small targets (0.25 high × 0.5 m wide) which show promise for cost-efficient control of Palpalis group tsetse flies. Consequently the performance of a small target was investigated for Glossina fuscipes fuscipes in Kenya, when the target was obscured following the placement of vegetation to simulate various degrees of natural bush encroachment. Catches decreased significantly only when the target was obscured by more than 80%. Even if a small target is underneath a very low overhanging bush (0.5 m above ground), the numbers of G. f. fuscipes decreased by only about 30% compared to a target in the open. We show that the efficiency of the small targets, even in small (1 m diameter) clearings, is largely uncompromised by vegetation re-growth because G. f. fuscipes readily enter between and under vegetation. The essential characteristic is that there should be some openings between vegetation. This implies that for this important vector of HAT, and possibly other Palpalis group flies, a smaller initial clearance zone around targets can be made and longer interval between site maintenance visits is possible both of which will result in cost savings for large scale operations. We also investigated and discuss other site features e.g. large solid objects and position in relation to the water's edge in terms of the efficacy of the small targets.

  18. CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

    PubMed

    Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

    2016-07-07

    Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

  19. Cryostat including heater to heat a target

    DOEpatents

    Pehl, R.H.; Madden, N.W.; Malone, D.F.

    1990-09-11

    A cryostat is provided which comprises a vacuum vessel; a target disposed within the vacuum vessel; a heat sink disposed within the vacuum vessel for absorbing heat from the detector; a cooling mechanism for cooling the heat sink; a cryoabsorption mechanism for cryoabsorbing residual gas within the vacuum vessel; and a heater for maintaining the target above a temperature at which the residual gas is cryoabsorbed in the course of cryoabsorption of the residual gas by the cryoabsorption mechanism. 2 figs.

  20. Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells

    PubMed Central

    Prakash, Satya; Malgorzata Urbanska, Aleksandra

    2008-01-01

    There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review. PMID:19707368

  1. 76 FR 175 - Faurecia Emissions Control Technologies Including On-Site Leased Workers From Adecco Employment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-03

    ... Technologies Including On-Site Leased Workers From Adecco Employment Servcies and Emcon Technologies, Troy, MI..., applicable to workers of Faurecia Emissions Control Technologies, Troy, Michigan, including on-site leased workers from Adecco Employment Services, Troy, Michigan. The Department's notice of determination was...

  2. 76 FR 54793 - Westpoint Home, Inc., Manufacturing Division, Including On-Site Leased Workers from Manpower...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-02

    ... Home, Inc., Manufacturing Division, Including On-Site Leased Workers from Manpower, Greenville, AL; Westpoint Home, Inc., Distribution Center, Including On-Site Leased Workers from Manpower, Greenville, AL..., applicable to workers of WestPoint Home, Inc., Manufacturing Division, Greenville, Alabama and WestPoint Home...

  3. 78 FR 28642 - Eastman Kodak Company, Electrographic Print Solutions, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-15

    ... Kodak Company, Electrographic Print Solutions, Including On-Site Leased Workers From Adecco and Datrose, Spencerport, New York; Eastman Kodak Company, IPS, Including On-Site Leased Workers From Adecco, Dayton, Ohio... Trade Adjustment Assistance (TAA) filed on behalf of Eastman Kodak Company, Electrographic Print...

  4. Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

    PubMed

    Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

    2018-01-02

    Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

  5. Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites.

    PubMed

    Wong, Michael H L; Bryan, Holly K; Copple, Ian M; Jenkins, Rosalind E; Chiu, Pak Him; Bibby, Jaclyn; Berry, Neil G; Kitteringham, Neil R; Goldring, Christopher E; O'Neill, Paul M; Park, B Kevin

    2016-03-24

    Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro. We show that one of these analogues, CDDO-epoxide (13), is comparable to 4 in terms of cytotoxicity and potency toward Nrf2 in rat hepatoma cells and stably modifies specific cysteine residues (namely, Cys-257, -273, -288, -434, -489, and -613) within Keap1, the major repressor of Nrf2, both in vitro and in living cells. Supported by molecular modeling, these data demonstrate the value of 13 for identifying site(s) of interaction with pharmacologically relevant targets and informing the continuing development of triterpenoids as novel drug candidates.

  6. 75 FR 62423 - Barnstead Thermolyne Corporation, a Subsidiary of Thermo Fisher Scientific, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-08

    ... related to the production of scientific laboratory equipment. New information shows that workers leased... Corporation, a Subsidiary of Thermo Fisher Scientific, Including On-Site Leased Workers From Sedona Staffing... Scientific, including on- site leased workers from Sedona Staffing, Dubuque, Iowa. The notice was published...

  7. 76 FR 22729 - Polaris Industries, Including On-Site Leased Workers From Westaff, Supply Technologies, Aerotek...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-22

    ..., Including On-Site Leased Workers From Westaff, Supply Technologies, Aerotek, Securitas Security Services..., 2011 to include on-site leased workers from Supply Technologies, Aerotek and Securitas Security Services. The notice was published in the Federal Register on December 13, 2010 (75 FR 77666) and February...

  8. Genetic variation in IL-16 miRNA target site and time to prostate cancer diagnosis in African American men

    PubMed Central

    Hughes, Lucinda; Ruth, Karen; Rebbeck, Timothy R.; Giri, Veda N.

    2013-01-01

    Background Men with a family history of prostate cancer and African American men are at high risk for prostate cancer and in need of personalized risk estimates to inform screening decisions. This study evaluated genetic variants in genes encoding microRNA (miRNA) binding sites for informing of time to prostate cancer diagnosis among ethnically-diverse, high-risk men undergoing prostate cancer screening. Methods The Prostate Cancer Risk Assessment Program (PRAP) is a longitudinal screening program for high-risk men. Eligibility includes men ages 35-69 with a family history of prostate cancer or African descent. Participants with ≥ 1 follow-up visit were included in the analyses (n=477). Genetic variants in regions encoding miRNA binding sites in four target genes (ALOX15, IL-16, IL-18, and RAF1) previously implicated in prostate cancer development were evaluated. Genotyping methods included Taqman® SNP Genotyping Assay (Applied Biosystems) or pyrosequencing. Cox models were used to assess time to prostate cancer diagnosis by risk genotype. Results Among 256 African Americans with ≥ one follow-up visit, the TT genotype at rs1131445 in IL-16 was significantly associated with earlier time to prostate cancer diagnosis vs. the CC/CT genotypes (p=0.013), with a suggestive association after correction for false-discovery (p=0.065). Hazard ratio after controlling for age and PSA for TT vs. CC/CT among African Americans was 3.0 (95% CI 1.26-7.12). No association to time to diagnosis was detected among Caucasians by IL-16 genotype. No association to time to prostate cancer diagnosis was found for the other miRNA target genotypes. Conclusions Genetic variation in IL-16 encoding miRNA target site may be informative of time to prostate cancer diagnosis among African American men enrolled in prostate cancer risk assessment, which may inform individualized prostate cancer screening strategies in the future. PMID:24061634

  9. 77 FR 62535 - Hydro Aluminum North America, Inc., Midwest Region, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-15

    ... Aluminum North America, Inc., Midwest Region, Including On- Site Leased Workers From Employment Group, Aerotek, and Manpower, Kalamazoo, Michigan; Hydro Aluminum North America, Inc., Midwest Region, Including... Aluminum North America, Inc., Kalamazoo, Michigan. The subject worker group includes on-site leased workers...

  10. The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

    PubMed

    Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

    2017-08-23

    The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

  11. 76 FR 27366 - CEVA Freight, LLC, Dell Logistics Division, Including On-Site Leased Workers From Prologistix...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-11

    ... Logistics Division, Including On-Site Leased Workers From Prologistix, Including Workers Whose Unemployment..., applicable to workers of CEVA Freight, LLC, Dell Logistics Division, including on-site leased workers from... workers employed at CEVA Freight, LLC, Dell Logistics Division, Winston-Salem, North Carolina who were...

  12. Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

    PubMed

    Flyak, Andrew I; Kuzmina, Natalia; Murin, Charles D; Bryan, Christopher; Davidson, Edgar; Gilchuk, Pavlo; Gulka, Christopher P; Ilinykh, Philipp A; Shen, Xiaoli; Huang, Kai; Ramanathan, Palaniappan; Turner, Hannah; Fusco, Marnie L; Lampley, Rebecca; Kose, Nurgun; King, Hannah; Sapparapu, Gopal; Doranz, Benjamin J; Ksiazek, Thomas G; Wright, David W; Saphire, Erica Ollmann; Ward, Andrew B; Bukreyev, Alexander; Crowe, James E

    2018-05-07

    Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs) 3-6 that target EBOV-specific epitopes on the surface glycoprotein 7,8 . However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

  13. Protecting Important Sites for Biodiversity Contributes to Meeting Global Conservation Targets

    PubMed Central

    Butchart, Stuart H. M.; Scharlemann, Jörn P. W.; Evans, Mike I.; Quader, Suhel; Aricò, Salvatore; Arinaitwe, Julius; Balman, Mark; Bennun, Leon A.; Bertzky, Bastian; Besançon, Charles; Boucher, Timothy M.; Brooks, Thomas M.; Burfield, Ian J.; Burgess, Neil D.; Chan, Simba; Clay, Rob P.; Crosby, Mike J.; Davidson, Nicholas C.; De Silva, Naamal; Devenish, Christian; Dutson, Guy C. L.; Fernández, David F. Día z; Fishpool, Lincoln D. C.; Fitzgerald, Claire; Foster, Matt; Heath, Melanie F.; Hockings, Marc; Hoffmann, Michael; Knox, David; Larsen, Frank W.; Lamoreux, John F.; Loucks, Colby; May, Ian; Millett, James; Molloy, Dominic; Morling, Paul; Parr, Mike; Ricketts, Taylor H.; Seddon, Nathalie; Skolnik, Benjamin; Stuart, Simon N.; Upgren, Amy; Woodley, Stephen

    2012-01-01

    Protected areas (PAs) are a cornerstone of conservation efforts and now cover nearly 13% of the world's land surface, with the world's governments committed to expand this to 17%. However, as biodiversity continues to decline, the effectiveness of PAs in reducing the extinction risk of species remains largely untested. We analyzed PA coverage and trends in species' extinction risk at globally significant sites for conserving birds (10,993 Important Bird Areas, IBAs) and highly threatened vertebrates and conifers (588 Alliance for Zero Extinction sites, AZEs) (referred to collectively hereafter as ‘important sites’). Species occurring in important sites with greater PA coverage experienced smaller increases in extinction risk over recent decades: the increase was half as large for bird species with>50% of the IBAs at which they occur completely covered by PAs, and a third lower for birds, mammals and amphibians restricted to protected AZEs (compared with unprotected or partially protected sites). Globally, half of the important sites for biodiversity conservation remain unprotected (49% of IBAs, 51% of AZEs). While PA coverage of important sites has increased over time, the proportion of PA area covering important sites, as opposed to less important land, has declined (by 0.45–1.14% annually since 1950 for IBAs and 0.79–1.49% annually for AZEs). Thus, while appropriately located PAs may slow the rate at which species are driven towards extinction, recent PA network expansion has under-represented important sites. We conclude that better targeted expansion of PA networks would help to improve biodiversity trends. PMID:22457717

  14. A multi-criteria targeting approach to neutral grassland conservation.

    PubMed

    Bayliss, Julian; Helyar, Alice; Lee, John T; Thompson, Stewart

    2003-02-01

    Resources for creating and managing rare habitats are limited, and a targeting approach aimed at identifying the most viable sites for habitat conservation is therefore desirable. This study developed a multi-criteria targeting approach to site conservation for two rare grassland types, based on a suite of biotic and abiotic factors managed within a Geographical Information System. A number of biotic and abiotic criteria were assessed to evaluate the biodiversity status of grassland sites. Biotic factors included species diversity, species richness and species rarity; and abiotic factors included patch area, position in the ecological unit and the influence of surrounding land use. Each criterion was given equal weighting and a final biodiversity value for each patch was calculated; the patch with the highest cumulative rank score was deemed the patch with the greatest biodiversity. Each site was then examined in relation to agricultural land under the existing management prescriptions of the Upper Thames Tributaries Environmentally Sensitive Area (UTTESA). Sites identified with high biodiversity potential, but currently not included under management prescriptions, were targeted for future inclusion in the ESA scheme. The targeting approach demonstrated how the national Lowland Meadows habitat action plan creation target of 500 ha could be achieved in the UTTESA. The fact that this target figure was so easily attained within this study area highlighted the possible underestimation of national habitat creation targets.

  15. Novel therapeutic approaches for pulmonary arterial hypertension: Unique molecular targets to site-specific drug delivery.

    PubMed

    Vaidya, Bhuvaneshwar; Gupta, Vivek

    2015-08-10

    Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by increased blood pressure in the small arterioles supplying blood to lungs for oxygenation. Advances in understanding of molecular and cellular biology techniques have led to the findings that PAH is indeed a cascade of diseases exploiting multi-faceted complex pathophysiology, with cellular proliferation and vascular remodeling being the key pathogenic events along with several cellular pathways involved. While current therapies for PAH do provide for amelioration of disease symptoms and acute survival benefits, their full therapeutic potential is hindered by patient incompliance and off-target side effects. To overcome the issues related with current therapy and to devise a more selective therapy, various novel pathways are being investigated for PAH treatment. In addition, inability to deliver anti-PAH drugs to the disease site i.e., distal pulmonary arterioles has been one of the major challenges in achieving improved patient outcomes and improved therapeutic efficacy. Several novel carriers have been explored to increase the selectivity of currently approved anti-PAH drugs and to act as suitable carriers for the delivery of investigational drugs. In the present review, we have discussed potential of various novel molecular pathways/targets including RhoA/Rho kinase, tyrosine kinase, endothelial progenitor cells, vasoactive intestinal peptide, and miRNA in PAH therapeutics. We have also discussed various techniques for site-specific drug delivery of anti-PAH therapeutics so as to improve the efficacy of approved and investigational drugs. This review will provide gainful insights into current advances in PAH therapeutics with an emphasis on site-specific drug payload delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

    DTIC Science & Technology

    2015-11-01

    increased PhScN potency as a result of preventing endoproteolytic degradation. Finally, the in vivo lung extravasation and colonization data, as well as...successful colonization are late stages in breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony...Award Number: TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma

  17. 75 FR 77664 - Honeywell International, Inc., Automation and Control Solutions Division, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-13

    ..., Inc., Automation and Control Solutions Division, Including On-Site Leased Workers From Manpower...., Automation and Control Solutions Division, Rock Island, Illinois. The notice was published in the Federal...-site at the Rock Island, Illinois location of Honeywell International, Inc., Automation and Control...

  18. 77 FR 4368 - Abbott Laboratories, Diagnostics Division, Including On-Site Leased Workers From Manpower...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-27

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-75,201] Abbott Laboratories..., applicable to workers of Abbott Laboratories, Diagnostics Division, including on-site leased workers from... (clerical) were employed on-site at the Irving, Texas location of Abbott Laboratories, Diagnostics Division...

  19. Outreach for Outreach: Targeting social media audiences to promote a NASA kids’ web site

    NASA Astrophysics Data System (ADS)

    Pham, C. C.

    2009-12-01

    The Space Place is a successful NASA web site that benefits upper elementary school students and educators by providing games, activities, and resources to stimulate interest in science, technology, engineering, and mathematics, as well as to inform the audience of NASA’s contributions. As online social networking grows to be a central component of modern communication, The Space Place has explored the benefits of integrating social networks with the web site to increase awareness of materials the web site offers. This study analyzes the capabilities of social networks, and specifically the demographics of Twitter and Facebook. It then compares these results with the content, audience, and perceived demographics of The Space Place web site. Based upon the demographic results, we identified a target constituency that would benefit from the integration of social networks into The Space Place web site. As a result of this study, a Twitter feed has been established that releases a daily tweet from The Space Place. In addition, a Facebook page has been created to showcase new content and prompt interaction among fans of The Space Place. Currently, plans are under way to populate the Space Place Facebook page. Each social network has been utilized in an effort to spark excitement about the content on The Space Place, as well as to attract followers to the main NASA Space Place web site. To pursue this idea further, a plan has been developed to promote NASA Space Place’s social media tools among the target audience.

  20. Targeted Nanotechnology for Cancer Imaging

    PubMed Central

    Toy, Randall; Bauer, Lisa; Hoimes, Christopher; Ghaghada, Ketan B.; Karathanasis, Efstathios

    2014-01-01

    Targeted nanoparticle imaging agents provide many benefits and new opportunities to facilitate accurate diagnosis of cancer and significantly impact patient outcome. Due to the highly engineerable nature of nanotechnology, targeted nanoparticles exhibit significant advantages including increased contrast sensitivity, binding avidity and targeting specificity. Considering the various nanoparticle designs and their adjustable ability to target a specific site and generate detectable signals, nanoparticles can be optimally designed in terms of biophysical interactions (i.e., intravascular and interstitial transport) and biochemical interactions (i.e., targeting avidity towards cancer-related biomarkers) for site-specific detection of very distinct microenvironments. This review seeks to illustrate that the design of a nanoparticle dictates its in vivo journey and targeting of hard-to-reach cancer sites, facilitating early and accurate diagnosis and interrogation of the most aggressive forms of cancer. We will report various targeted nanoparticles for cancer imaging using X-ray computed tomography, ultrasound, magnetic resonance imaging, nuclear imaging and optical imaging. Finally, to realize the full potential of targeted nanotechnology for cancer imaging, we will describe the challenges and opportunities for the clinical translation and widespread adaptation of targeted nanoparticles imaging agents. PMID:25116445

  1. Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

    PubMed Central

    Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

    2013-01-01

    The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

  2. Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum.

    PubMed

    Dong, Hongyan; Yauk, Carole L; Rowan-Carroll, Andrea; You, Seo-Hee; Zoeller, R Thomas; Lambert, Iain; Wade, Michael G

    2009-01-01

    Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

  3. Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

    DTIC Science & Technology

    2015-11-01

    systemic therapy to prevent breast cancer bone colony progression. Figure 6. Colocalization of Ac-PhscNGGK-Bio with DiI in lung– extravasated SUM149PT cells...breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony formation would increase life...1 Award Number: W81XWH-12-1-0097 TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site

  4. 75 FR 26793 - Beam Global Spirits & Wine, Including On-Site Leased Workers from Adecco, St. Elizabeth Business...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... & Wine, Including On-Site Leased Workers from Adecco, St. Elizabeth Business Health, Guardsmark, and Lab... 26, 2010, applicable to workers of Beam Global Spirits & Wine, including on-site leased workers from... on-site at the Cincinnati, Ohio location of Beam Global Spirits & Wine. The Department has determined...

  5. Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

    PubMed Central

    Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

    2009-01-01

    Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

  6. 75 FR 39051 - Allstate Insurance Company, Altoona Express Market Claim Office, Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-07

    ... former workers of Allstate Insurance Company, Altoona Express Market Claim Office, including on- site... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,536] Allstate Insurance Company, Altoona Express Market Claim Office, Including On-Site Leased Workers From Kelly Services...

  7. De-novo discovery of differentially abundant transcription factor binding sites including their positional preference.

    PubMed

    Keilwagen, Jens; Grau, Jan; Paponov, Ivan A; Posch, Stefan; Strickert, Marc; Grosse, Ivo

    2011-02-10

    Transcription factors are a main component of gene regulation as they activate or repress gene expression by binding to specific binding sites in promoters. The de-novo discovery of transcription factor binding sites in target regions obtained by wet-lab experiments is a challenging problem in computational biology, which has not been fully solved yet. Here, we present a de-novo motif discovery tool called Dispom for finding differentially abundant transcription factor binding sites that models existing positional preferences of binding sites and adjusts the length of the motif in the learning process. Evaluating Dispom, we find that its prediction performance is superior to existing tools for de-novo motif discovery for 18 benchmark data sets with planted binding sites, and for a metazoan compendium based on experimental data from micro-array, ChIP-chip, ChIP-DSL, and DamID as well as Gene Ontology data. Finally, we apply Dispom to find binding sites differentially abundant in promoters of auxin-responsive genes extracted from Arabidopsis thaliana microarray data, and we find a motif that can be interpreted as a refined auxin responsive element predominately positioned in the 250-bp region upstream of the transcription start site. Using an independent data set of auxin-responsive genes, we find in genome-wide predictions that the refined motif is more specific for auxin-responsive genes than the canonical auxin-responsive element. In general, Dispom can be used to find differentially abundant motifs in sequences of any origin. However, the positional distribution learned by Dispom is especially beneficial if all sequences are aligned to some anchor point like the transcription start site in case of promoter sequences. We demonstrate that the combination of searching for differentially abundant motifs and inferring a position distribution from the data is beneficial for de-novo motif discovery. Hence, we make the tool freely available as a component of the open

  8. 76 FR 2145 - International Paper Company, Franklin Pulp & Paper Mill, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-12

    ... Company, Franklin Pulp & Paper Mill, Including On-Site Leased Workers From Railserve, Franklin, VA..., applicable to workers and former workers of International Paper Company, Franklin Pulp & Paper Mill, Franklin... follows: All workers International Paper Company, Franklin Pulp & Paper Mill, including on-site leased...

  9. 75 FR 76037 - General Motors Corporation Grand Rapids Metal Center Metal Fabricating Division Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... Corporation Grand Rapids Metal Center Metal Fabricating Division Including On-Site Leased Workers From... Corporation, Grand Rapids Metal Center, Metal Fabricating Division, including on- site leased workers from... of metal stampings and sub- assembled metal sheet components. The company reports that workers leased...

  10. 78 FR 70580 - Amphenol Backplane Systems, Including On-Site Leased Workers From Technical Needs and National...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-26

    ... Systems, Including On-Site Leased Workers From Technical Needs and National Engineering, Nashua, New... to the production of electrical connectors and backplane assemblies. The subject worker group includes on-site leased workers from Technical Needs and National Engineering. Workers of the subject firm...

  11. 75 FR 11921 - Heritage Aviation, Ltd., Including On-Site Leased Workers From Global Technical Services and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,924] Heritage Aviation, Ltd., Including On-Site Leased Workers From Global Technical Services and Global, Inc. (Global Employment... from Heritage Aviation, Ltd, including on-site leased workers from Global Technical Services, Grand...

  12. 75 FR 45159 - Stanadyne Corporation, Including On-Site Leased Workers From Infini-Staff, Staffing Now and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ..., Including On-Site Leased Workers From Infini-Staff, Staffing Now and Apollo Professional Solutions, Inc... shows that workers leased from Apollo Professional Solutions were employed on-site at the Windsor..., the Department is amending this certification to include workers leased from Apollo Professional...

  13. 77 FR 14832 - Plumchoice, Inc., Including On-Site Leased Workers From Balance Staffing, Insight Global Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-13

    ...., Including On-Site Leased Workers From Balance Staffing, Insight Global Staffing, and Technisource..., 2012, applicable to workers of PlumChoice, Inc., including on-site leased workers from Balance Staffing... leased workers from Balance Staffing, Insight Global Staffing, and Technisource, Scarborough, Maine, who...

  14. 77 FR 44685 - ConocoPhillips Company, Trainer Refinery, Including On-Site Leased Workers From Shrack, Young...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-30

    ..., Trainer Refinery, Including On-Site Leased Workers From Shrack, Young, and Associates, Inc., and Project... applicable to workers and former workers of ConocoPhillips Company, Trainer Refinery, Trainer, Pennsylvania..., and heavy oil. The subject worker group includes on-site leased workers from Shrack, Young, and...

  15. 75 FR 11913 - Chrysler, LLC; Warren Stamping Plant, Including On-Site Leased Workers From Caravan Knight...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... Stamping Plant, Including On-Site Leased Workers From Caravan Knight Facilities Management LLC; Warren, MI... from Caravan Knight Facilities Management LLC were employed on-site at the Warren, Michigan location of... Department is amending this certification to include workers leased from Caravan Knight Facilities Management...

  16. 75 FR 41523 - Paris Accessories, Inc., Including On-Site Leased Workers From Job Connections, New Smithville...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-16

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,106; TA-W-71,106A] Paris Accessories, Inc., Including On-Site Leased Workers From Job Connections, New Smithville, PA; Paris... Paris Accessories, Inc., including on-site leased workers from Job Connections, New Smithville...

  17. 78 FR 15049 - The Evercare Company Including On-Site Leased Workers From Global Personnel Solutions and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-08

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,932] The Evercare Company Including On-Site Leased Workers From Global Personnel Solutions and Manpower Waynesboro, Georgia; Amended... group included on-site leased workers from Global Personnel Solutions. In response to an inquiry by the...

  18. 76 FR 50272 - West, A Thomson Reuters Business, Thomson Reuters Legal Division, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-75,099] West, A Thomson Reuters Business, Thomson Reuters Legal Division, Including On-Site Leased Workers From Adecco, Albuquerque, New... former workers of West, A Thomson Reuters Business, Thomson Reuters Legal Division, including On-Site...

  19. 75 FR 16514 - Bayer Material Science, LLC, Formally Known as Sheffield Plastics, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-01

    ..., LLC, Formally Known as Sheffield Plastics, Including On-Site Leased Workers from Randstadt Work... Plastics, including on-site leased workers from Randstadt Work Solutions, Berlin, Connecticut. The notice... Plastics. Some workers separated from employment at the subject firm had their wages reported under two...

  20. The ATP-binding site of type II topoisomerases as a target for antibacterial drugs.

    PubMed

    Maxwell, Anthony; Lawson, David M

    2003-01-01

    DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

  1. 75 FR 76038 - Zach System Corporation a Subdivision of Zambon Company, SPA Including On-Site Leased Workers of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... Subdivision of Zambon Company, SPA Including On-Site Leased Workers of Turner Industries and Go Johnson, La..., including on-site leased workers from Turner Industries and Go Johnson, La Porte, Texas. The Department's..., including on-site leased workers of Turner Industries and Go Johnson, La Porte, Texas, who became totally or...

  2. Occurrence Prospect of HDR and Target Site Selection Study in Southeastern of China

    NASA Astrophysics Data System (ADS)

    Lin, W.; Gan, H.

    2017-12-01

    Hot dry rock (HDR) geothermal resource is one of the most important clean energy in future. Site selection a HDR resource is a fundamental work to explore the HDR resources. This paper compiled all the HDR development projects domestic and abroad, and summarized the location of HDR geothermal geological index. After comparing the geological background of HDR in the southeast coastal area of China, Yangjiang Xinzhou in Guangdong province, Leizhou Peninsula area, Lingshui in Hainan province and Huangshadong in Guangzhou were selected from some key potential target area along the southeast coast of China. Deep geothermal field model of the study area is established based on the comprehensive analysis of the target area of deep geothermal geological background and deep thermal anomalies. This paper also compared the hot dry rock resources target locations, and proposed suggestions for the priority exploration target area and exploration scheme.

  3. 77 FR 3499 - Sony Electronics, Inc., Including On-Site Leased Workers From Selectremedy Park Ridge, NJ...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,501M] Sony Electronics, Inc... Electronics, Inc., SEL Headquarters, including on-site leased workers of SelectRemedy, StaffMark, and Payrolling.com , San Diego, California (TA-W-71,501); Sony Electronics, Inc., including on-site leased...

  4. 78 FR 61389 - Sanyo Solar of Oregon, LLC, Wafer Slicing and Quality Control Operations, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-03

    ..., LLC, Wafer Slicing and Quality Control Operations, Including On-Site Leased Workers From Brown and... Quality Control Operations, Salem, Oregon, including on-site leased workers from Brown and Dunton, Inc... and included workers who supplied quality control and support functions. The company reports that...

  5. Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

    PubMed Central

    Alcántara-de la Cruz, Ricardo; Fernández-Moreno, Pablo T.; Ozuna, Carmen V.; Rojano-Delgado, Antonia M.; Cruz-Hipolito, Hugo E.; Domínguez-Valenzuela, José A.; Barro, Francisco; De Prado, Rafael

    2016-01-01

    In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world. PMID:27752259

  6. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

    PubMed Central

    Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

    1992-01-01

    opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

  7. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

    PubMed

    Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F

    2017-03-07

    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

  8. 76 FR 2709 - Whirlpool Corporation, Benton Harbor Division, Including On-Site Leased Workers of Aerotek and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-14

    ..., Benton Harbor Division, Including On-Site Leased Workers of Aerotek and Penske Logistics, LLC, Benton... equipment. The company reports that workers leased from Penske Logistics, LLC, were employed on-site at the... from Penske Logistics, LLC working on-site at the Benton Harbor, Michigan location of Whirlpool...

  9. Bombing Target Identification from Limited Transect Data

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

    Roberts, Barry L.; Hathaway, John E.; Pulsipher, Brent A.

    2006-08-07

    A series of sensor data combined with geostatistical techniques were used to determine likely target areas for a historic military aerial bombing range. Primary data consisted of magnetic anomaly information from limited magnetometer transects across the site. Secondary data included airborne LIDAR, orthophotography, and other general site characterization information. Identification of likely target areas relied primarily upon kriging estimates of magnetic anomaly densities across the site. Secondary information, such as impact crater locations, was used to refine the boundary delineations.

  10. TAPIR, a web server for the prediction of plant microRNA targets, including target mimics.

    PubMed

    Bonnet, Eric; He, Ying; Billiau, Kenny; Van de Peer, Yves

    2010-06-15

    We present a new web server called TAPIR, designed for the prediction of plant microRNA targets. The server offers the possibility to search for plant miRNA targets using a fast and a precise algorithm. The precise option is much slower but guarantees to find less perfectly paired miRNA-target duplexes. Furthermore, the precise option allows the prediction of target mimics, which are characterized by a miRNA-target duplex having a large loop, making them undetectable by traditional tools. The TAPIR web server can be accessed at: http://bioinformatics.psb.ugent.be/webtools/tapir. Supplementary data are available at Bioinformatics online.

  11. 78 FR 48468 - Delphi Corporation, Electronics and Safety Division, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ..., Electronics and Safety Division, Including On-Site Leased Workers From Securitas, Bartech, Flint Janitorial... Adjustment Assistance on May 20, 2013, applicable to workers of Delphi Corporation, Electronics and Safety... on- site at the Flint, Michigan location of Delphi Corporation, Electronics and Safety Division. The...

  12. 76 FR 72978 - Whirlpool Corporation Including On-Site Leased Workers From Career Solutions TEC Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ..., TEK Systems, Penske Logistics, Eurest, and Canteen, Fort Smith, AR; Amended Certification Regarding... Corporation, including on-site leased workers from Career Solutions TEC Staffing, Fort Smith, Arkansas. The... Corporation, TEK Systems, Penske Logistics, Eurest, and Canteen were employed on-site at the Fort Smith...

  13. 75 FR 20386 - Trane a Subsidiary of Ingersoll Rand Including On-Site Leased Workers From Aerotek, Express...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-19

    ... Ingersoll Rand Including On-Site Leased Workers From Aerotek, Express Personnel Services, Select Staffing... Ingersoll Rand, including on-site leased workers from Aerotek, Express Personnel Staffing, and Select... from Aerotek, Express Personnel Staffing, Select Staffing, and Mechanical Contractors, Inc., Pueblo...

  14. 78 FR 28633 - Prometric, Inc., a Subsidiary of Educational Testing Service, Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-15

    ... Subsidiary of Educational Testing Service, Including On-Site Leased Workers From Office Team St. Paul... Office Team were employed on-site at the St. Paul, Minnesota location of the subject firm. The Department... workers leased from Office Team working on- site at the St. Paul, Minnesota location of Prometric, Inc., a...

  15. 75 FR 3250 - Glaxosmithkline, Including On-Site Temporary and Leased Workers From Kelly Services, Kelly...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-20

    ... Dream Clean, Inc., Bristol, TN; Amended Certification Regarding Eligibility To Apply for Worker... animals. The company reports that on-site leased workers from Dream Clean, Inc. were employed on-site at... findings, the Department is amending this certification to include workers leased from Dream Clean, Inc...

  16. 75 FR 38128 - Ceva Freight, LLC, Dell Logistics Division, Including On-Site Leased Workers From Prologistix and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-01

    ... Logistics Division, Including On-Site Leased Workers From Prologistix and Employment Staffing Solutions... 19, 2010, applicable to workers of CEVA Freight, LLC, Dell Logistics Division, including on-site..., North Carolina location of CEVA Freight, LLC, Dell Logistics Division. The Department has determined...

  17. 76 FR 66328 - Callaway Golf Ball Operations, Inc., Including On-Site Leased Workers From Reliable Temp Services...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-26

    ... Operations, Inc., Including On-Site Leased Workers From Reliable Temp Services, Inc., Johnson & Hill Staffing... Golf Ball Operations, Inc., including on-site leased workers from Reliable Temp Services, Inc., and... Reliable Temp Services, Inc., Johnson & Hill Staffing and Apollo Security, Chicopee, Massachusetts, who...

  18. 75 FR 20389 - Chrysler Group LLC, Formerly Known as Chrysler LLC, Warren Office Building, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-19

    ... certification to include workers leased from Product Action International, LLC working on-site at the Warren..., Formerly Known as Chrysler LLC, Warren Office Building, Including On-Site Leased Workers from Product Action International, LLC, Warren, Michigan; Amended Certification Regarding Eligibility To Apply for...

  19. 78 FR 21155 - Eastman Kodak Company, IPS-Dayton Location, Including On-site Leased Workers From Adecco, Dayton...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-09

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,387] Eastman Kodak Company, IPS--Dayton Location, Including On-site Leased Workers From Adecco, Dayton, Ohio; Notice of... and former workers of Eastman Kodak Company, IPS--Dayton Location, including on-site leased workers...

  20. 75 FR 45158 - Holcim (US) Inc. Corporate Division Including On-Site Leased Workers From Manpower, Office Team...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    .... Corporate Division Including On-Site Leased Workers From Manpower, Office Team and Advance Temporary... from Manpower and Office Team, Dundee, Michigan. The notice was published in the Federal Register on... Holcim (US) Inc., Corporate Division, including on-site leased workers from Manpower, Office Team Advance...

  1. 77 FR 12081 - Keithley Instruments Including On-Site Leased Workers from StaffMatrix and ADECCO, Solon, OH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-28

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-80,264] Keithley Instruments Including On-Site Leased Workers from StaffMatrix and ADECCO, Solon, OH; Amended Certification Regarding... Instruments, including on-site leased workers from StaffMatrix and Adecco, Solon, Ohio, who became totally or...

  2. Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

    PubMed Central

    Ding, Kejia; Wang, Annie; Boerneke, Mark A.; Dibrov, Sergey M.; Hermann, Thomas

    2014-01-01

    We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex. PMID:24856063

  3. Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.

    PubMed

    Kakimoto, Yuriko; Tashiro, Shinya; Kojima, Rieko; Morozumi, Yuki; Endo, Toshiya; Tamura, Yasushi

    2018-04-18

    Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

  4. Binding site and affinity prediction of general anesthetics to protein targets using docking.

    PubMed

    Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

    2012-05-01

    The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site

  5. Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

    PubMed Central

    Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

    2012-01-01

    Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the

  6. 75 FR 5147 - Acushnet Company a Subsidiary of Fortune Brands Including On-Site Leased Workers From Olsten...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-01

    ...,763B] Acushnet Company a Subsidiary of Fortune Brands Including On-Site Leased Workers From Olsten Staffing Services Fairhaven, MA; Acushnet Company a Subsidiary of Fortune Brands Including On-Site Leased Workers From Olsten Staffing Services New Bedford, MA; Acushnet Company a Subsidiary of Fortune Brands...

  7. 75 FR 10318 - Venta-Airwasher, LLC, Including On-Site Leased Workers From Metro Staffing, Snelling and Office...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-05

    ..., Including On-Site Leased Workers From Metro Staffing, Snelling and Office Team Itasca, IL; Amended... from Metro Staffing, Snelling and Office Team were employed on-site at the Itasca, Illinois location of... amending this certification to include workers leased from Metro Staffing, Snelling and Office Team working...

  8. Site-Specific Targeting of Platelet-Rich Plasma via Superparamagnetic Nanoparticles

    PubMed Central

    Talaie, Tara; Pratt, Stephen J.P.; Vanegas, Camilo; Xu, Su; Henn, R. Frank; Yarowsky, Paul; Lovering, Richard M.

    2015-01-01

    Background: Muscle strains are one of the most common injuries treated by physicians. Standard conservative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus regarding treatments to accelerate recovery. Recently, clinical use of platelet-rich plasma (PRP) has gained momentum as an option for therapy and is appealing for many reasons, most notably because it provides growth factors in physiological proportions and it is autologous, safe, easily accessible, and potentially beneficial. Local delivery of PRP to injured muscles can hasten recovery of function. However, specific targeting of PRP to sites of tissue damage in vivo is a major challenge that can limit its efficacy. Hypothesis: Location of PRP delivery can be monitored and controlled in vivo with noninvasive tools. Study Design: Controlled laboratory study. Methods: Superparamagnetic iron oxide nanoparticles (SPIONs) can be visualized by both magnetic resonance imaging (MRI) (in vivo) and fluorescence microscopy (after tissue harvesting). PRP was labeled with SPIONs and administered by intramuscular injections of SPION-containing platelets. MRI was used to monitor the ability to manipulate and retain the location of PRP in vivo by placement of an external magnet. Platelets were isolated from whole blood and incubated with SPIONs. Following SPION incubation with PRP, a magnetic field was used to manipulate platelet location in culture dishes. In vivo, the tibialis anterior (TA) muscles of anesthetized Sprague-Dawley rats were injected with SPION-containing platelets, and MRI was used to track platelet position with and without a magnet worn over the TA muscles for 4 days. Results: The method used to isolate PRP yielded a high concentration (almost 4-fold increase) of platelets. In vitro experiments showed that the platelets successfully took up SPIONs and then rapidly responded to an applied magnetic field

  9. 76 FR 13662 - Pass & Seymour/Legrand a Subsidiary of Legrand North America; Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-14

    ... Subsidiary of Legrand North America; Including On-Site Leased Workers From Select Staffing, also Known as... subsidiary of Legrand North America, including on-site leased workers from Select Staffing and Aerotek... reviewed the certification for workers of the subject firm. The company reports that Select Staffing, an on...

  10. Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation

    PubMed Central

    Albritton, Sarah Elizabeth; Kranz, Anna-Lena; Winterkorn, Lara Heermans; Street, Lena Annika; Ercan, Sevinc

    2017-01-01

    In many organisms, it remains unclear how X chromosomes are specified for dosage compensation, since DNA sequence motifs shown to be important for dosage compensation complex (DCC) recruitment are themselves not X-specific. Here, we addressed this problem in C. elegans. We found that the DCC recruiter, SDC-2, is required to maintain open chromatin at a small number of primary DCC recruitment sites, whose sequence and genomic context are X-specific. Along the X, primary recruitment sites are interspersed with secondary sites, whose function is X-dependent. A secondary site can ectopically recruit the DCC when additional recruitment sites are inserted either in tandem or at a distance (>30 kb). Deletion of a recruitment site on the X results in reduced DCC binding across several megabases surrounded by topologically associating domain (TAD) boundaries. Our work elucidates that hierarchy and long-distance cooperativity between gene-regulatory elements target a single chromosome for regulation. DOI: http://dx.doi.org/10.7554/eLife.23645.001 PMID:28562241

  11. Novel medical therapeutics in glioblastomas, including targeted molecular therapies, current and future clinical trials.

    PubMed

    Quant, Eudocia C; Wen, Patrick Y

    2010-08-01

    The prognosis for glioblastoma is poor despite optimal therapy with surgery, radiation, and chemotherapy. New therapies that improve survival and quality of life are needed. Research has increased our understanding of the molecular pathways important for gliomagenesis and disease progression. Novel agents have been developed against these targets, including receptor tyrosine kinases, intracellular signaling molecules, epigenetic abnormalities, and tumor vasculature and microenvironment. This article reviews novel therapies for glioblastoma, with an emphasis on targeted agents. Copyright 2010 Elsevier Inc. All rights reserved.

  12. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

    PubMed Central

    Kim, Min Jung; Chia, Ian V.; Costantini, Frank

    2008-01-01

    Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

  13. Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

    PubMed Central

    Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

    2012-01-01

    Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

  14. 75 FR 21352 - Chrysler Group LLC, Formerly Known as Chrysler LLC; Belvidere Assembly Plant: Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-23

    ..., Formerly Known as Chrysler LLC; Belvidere Assembly Plant: Including On-Site Leased Workers From Aerotek, G..., formerly known as Chrysler LLC, Belvidere Assembly Plant, include on- site leased workers from Aerotek and... certification for workers of the subject firm. The workers are engaged in activities related to the assembly the...

  15. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

  16. 77 FR 21588 - Hart and Cooley, Inc., A Subsidiary of Tomkins, PLC Including On-Site Leased Workers from...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-10

    ... Subsidiary of Tomkins, PLC Including On- Site Leased Workers from Reliable, Masiello Employment Services..., applicable to workers of Hart and Cooley, Inc., a subsidiary of Tomkins, PLC, including on-site leased... issued as follows: ``All workers from Hart and Cooley, Inc., a subsidiary of Tomkins, PLC, including on...

  17. LPT. Plot plan and site layout. Includes shield test pool/EBOR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    LPT. Plot plan and site layout. Includes shield test pool/EBOR facility. (TAN-645 and -646) low power test building (TAN-640 and -641), water storage tanks, guard house (TAN-642), pump house (TAN-644), driveways, well, chlorination building (TAN-643), septic system. Ralph M. Parsons 1229-12 ANP/GE-7-102. November 1956. Approved by INEEL Classification Office for public release. INEEL index code no. 038-0102-00-693-107261 - Idaho National Engineering Laboratory, Test Area North, Scoville, Butte County, ID

  18. Fitness cost implications of phiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae)

    USDA-ARS?s Scientific Manuscript database

    Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understandin...

  19. 76 FR 14691 - Delphi Steering, Including On-Site Leased Workers From Acro Service Corporation, Aerotek, Inc...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-17

    ... Health Care and Advantage Technical Resourcing, Saginaw, MI; Amended Certification Regarding Eligibility... on-site leased workers from Advantage Technical Resourcing were employed on-site at the Saginaw... Department is amending this certification to include workers leased from Advantage Technical Resourcing...

  20. 76 FR 7585 - General Motors Corporation Milford Proving Grounds Including On-Site Leased Workers From Adroit...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-10

    ... on-site leased workers from Adroit Software & Consulting, Inc., Aerotek Professional Services, Inc... Corporation Milford Proving Grounds Including On- Site Leased Workers From Adroit Software & Consulting, Inc., Aerotek Professional Services, Inc., Aerotek, Inc., Ajilon Consulting (IS&S), Altair Engineering, Inc...

  1. Targeted Delivery of LXR Agonist Using a Site-Specific Antibody-Drug Conjugate.

    PubMed

    Lim, Reyna K V; Yu, Shan; Cheng, Bo; Li, Sijia; Kim, Nam-Jung; Cao, Yu; Chi, Victor; Kim, Ji Young; Chatterjee, Arnab K; Schultz, Peter G; Tremblay, Matthew S; Kazane, Stephanie A

    2015-11-18

    Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50-27 nM), but had no significant effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher-fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3-fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

  2. 76 FR 73684 - Tiger Drylac USA, Inc., Including On-Site Leased Workers From Berks and Beyond Employment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-29

    ...., Including On-Site Leased Workers From Berks and Beyond Employment Services, Gage Personnel and Office Team... Employment Services, Gage Personnel, and Office Team/Robert Half International were employed on- site at the... Personnel, and Office Team/Robert Half International working on-site at the Reading, Pennsylvania location...

  3. 78 FR 8588 - Franklin Electric Company, Inc., Including On-Site Leased Workers From Peoplelink Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-06

    ..., Inc., Including On-Site Leased Workers From Peoplelink Staffing Solutions, Remedy Intelligent Staffing... the immediate certification. The Department has received information that workers from Remedy..., the Department is amending this certification to include workers leased from Remedy Intelligent...

  4. 77 FR 67399 - State Street Corporation, Putnam Cash Reconciliations Team, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-09

    ..., Putnam Cash Reconciliations Team, Including On-Site Leased Workers From APC Workforce Solutions II, LLC... Corporation, Putnam Cash Reconciliation Team, Quincy, Massachusetts. The workers were engaged in activities... employed on-site at State Street Corporation, Putnam Cash Reconciliation Team, Quincy, Massachusetts. The...

  5. Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies

    DTIC Science & Technology

    2016-10-01

    2016 4 . TITLE AND SUBTITLE 5a. CONTRACT NUMBER Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5b. GRANT NUMBER 5c...new biomarker driven trials directly to patients W81XWH-14-2-0159 None listed 20 Table of Contents Page 1. Introduction…………………………………………………………. 4 2...Keywords……………………………………………………………. 4 3. Accomplishments ..……..…………………………………………... 4 4 . Impact…………………………...…………………………………… 8 5. Changes/Problems

  6. 75 FR 76487 - Haldex Brake Corporation, Commercial Vehicle Systems, Including On-Site Leased Workers of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-08

    ..., Commercial Vehicle Systems, Including On-Site Leased Workers of Johnston Integration Technologies, a... system components. The company reports that workers leased from Johnston Integration Technologies, a... certification to include workers leased from Johnston Integration Technologies, a subsidiary of Johnston...

  7. 78 FR 8587 - Heraeus Kulzer, LLC., Including On-Site Leased Workers from People Link Staffing, Forge Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-06

    ...., Including On-Site Leased Workers from People Link Staffing, Forge Staffing, Career Transitions and Talent... Career Transitions and Talent Source were employed on-site at the South Bend, Indiana location of Heraeus... workers leased from Career Transitions and Talent Source working on-site at the South Bend, Indiana...

  8. Target organs in chronic bioassays of 533 chemical carcinogens

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

    Gold, L.S.; Slone, T.H.; Manley, N.B.

    1991-06-01

    A compendium of carcinogenesis bioassay results organized by target organ is presented for 533 chemicals that are carcinogenic in at least one species. This compendium is based primarily on experiments in rats or mice; results in hamsters, nonhuman primates, and dogs are also reported. The compendium can be used to identify chemicals that induce tumors at particular sites, and to determine whether target sites are the same for chemicals positive in more than one species. The Carcinogenic Potency Database (CPDB), which includes results of 3969 experiments, is used in the analysis. The published CPDB includes details on each test, andmore » literature references. Chemical carcinogens are reported for 35 different target organs in rats or mice. More than 80% of the carcinogens in each of these species are positive in at least one of the 8 most frequent target sites; liver, lung, mammary gland, stomach, vascular system, kidney, hematopoietic system, and urinary bladder. An analysis is presented of how well one can predict the carcinogenic response in mice from results in rats, or vice versa. Among chemicals tested in both species, 76% of rat carcinogens are positive in mice, and 71% of mouse carcinogens are positive in rats. Prediction is less accurate to the same target site: 52% of rat carcinogens are positive in the same site in mice, and 48% of mouse carcinogens are positive in the same site in rats. The liver is the most frequent site in common between rats and mice.« less

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

    PubMed Central

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

    2015-01-01

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

  10. The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription

    PubMed Central

    Jennings, Barbara H.

    2014-01-01

    Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in “active” chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation

  11. 75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-23

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,642] Intel Corporation, Fab 20... of Intel Corporation, Fab 20 Division, including on-site leased workers of Volt Technical Resources... Precision, Inc. were employed on-site at the Hillsboro, Oregon location of Intel Corporation, Fab 20...

  12. 75 FR 77665 - Whirlpool Corporation, Including On-Site Leased Workers From Career Solutions TEC Staffing and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-13

    ..., Including On-Site Leased Workers From Career Solutions TEC Staffing and Andrews International, Fort Smith... Staffing, Fort Smith, Arkansas. The notice was published in the Federal Register on October 25, 2010 (75 FR... reports that workers leased from Andrews International were employed on-site at the Fort Smith, Arkansas...

  13. 77 FR 44679 - Suntron Corporation, Including On-Site Leased Workers From Manpower, Nesco, TPI and Robert Half...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-30

    ... workers leased from NESCO, TPI, and Robert Half working on-site at the Sugarland, Texas location of the... Suntron Corporation, including on-site leased workers from Manpower, NESCO, TPI, and Robert Half, Sugar...

  14. 76 FR 39129 - Tensolite, LLC D/B/A Carlisle Interconnect Assemblies Including On-Site Leased Workers From Volt...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-05

    ... Carlisle Interconnect Assemblies Including On-Site Leased Workers From Volt Services Group and Adecco..., applicable to workers of Tensolite, LLC, d/b/a Carlisle Interconnect Assemblies, including on-site leased... interconnect assemblies. The notice was published in the Federal Register on September 2, 2009 (74 FR 45476...

  15. 77 FR 56870 - New Process Gear, a Division of Magna Powertrain, Including On-Site Leased Workers From ABM...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-14

    ... Process Gear, a division of Magna Powertrain, including on-site leased workers from ABM Janitorial Service... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,940] New Process Gear, a Division of Magna Powertrain, Including On- Site Leased Workers From ABM Janitorial Service Northeast, Inc...

  16. 78 FR 2287 - Daimler Buses North America, Inc., a Subsidiary of Daimler North America Corp, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-10

    ... subsidiary of Daimler North America Corp., including on-site leased workers from Noramtec, First Choice... America, Inc., a Subsidiary of Daimler North America Corp, Including On-Site Leased Workers From Noramtec, First Choice Staffing, Staff Works, and Mr. Santo Lamarco From Wurth Revcar Fasteners, Inc., Oriskany...

  17. Site-specific antibody-liposome conjugation through copper-free click chemistry: a molecular biology approach for targeted photodynamic therapy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Obaid, Girgis; Wang, Yucheng; Kuriakose, Jerrin; Broekgaarden, Mans; Alkhateeb, Ahmed; Bulin, Anne-Laure; Hui, James; Tsourkas, Andrew; Hasan, Tayyaba

    2016-03-01

    Nanocarriers, such as liposomes, have the ability to potentiate photodynamic therapy (PDT) treatment regimens by the encapsulation of high payloads of photosensitizers and enhance their passive delivery to tumors through the enhanced permeability and retention effect. By conjugating targeting moieties to the surface of the liposomal nanoconstructs, cellular selectivity is imparted on them and PDT-based therapies can be performed with significantly higher dose tolerances, as off-target toxicity is simultaneously reduced.1 However, the maximal benefits of conventional targeted nanocarriers, including liposomes, are hindered by practical limitations including chemical instability, non-selective conjugation chemistry, poor control over ligand orientation, and loss of ligand functionality following conjugation, amongst others.2 We have developed a robust, physically and chemically stable liposomal nanoplatform containing benzoporphyrin derivative photosensitizer molecules within the phospholipid bilayer and an optimized surface density of strained cyclooctyne moieties for `click' conjugation to azido-functionalized antibodies.3 The clinical chimeric anti-EGFR antibody Cetuximab is site-specifically photocrosslinked to a recombinant bioengineered that recognizes the antibody's Fc region, containing a terminal azide.4 The copper-free click conjugation of the bioengineered Cetuximab derivative to the optimized photosensitizing liposome provides exceptional control over the antibody's optimal orientation for cellular antigen binding. Importantly, the reaction occurs rapidly under physiological conditions, bioorthogonally (selectively in the presence of other biomolecules) and without the need for toxic copper catalysis.3 Such state-of-the-art conjugation strategies push the boundaries of targeted photodynamic therapy beyond the limitations of traditional chemical coupling techniques to produce more robust and effective targeted therapeutics with applications beyond

  18. 49 CFR 325.77 - Computation of open site requirements-nonstandard sites.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... microphone target point is other than 50 feet (15.2 m), the test site must be an open site within a radius... microphone target point. (b) Plan view diagrams of nonstandard test sites are shown in Figures 3 and 4... (18.3 m) distance between the microphone location point and the microphone target point. (See § 325.79...

  19. 49 CFR 325.77 - Computation of open site requirements-nonstandard sites.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... microphone target point is other than 50 feet (15.2 m), the test site must be an open site within a radius... microphone target point. (b) Plan view diagrams of nonstandard test sites are shown in Figures 3 and 4... (18.3 m) distance between the microphone location point and the microphone target point. (See § 325.79...

  20. TargetM6A: Identifying N6-Methyladenosine Sites From RNA Sequences via Position-Specific Nucleotide Propensities and a Support Vector Machine.

    PubMed

    Li, Guang-Qing; Liu, Zi; Shen, Hong-Bin; Yu, Dong-Jun

    2016-10-01

    As one of the most ubiquitous post-transcriptional modifications of RNA, N 6 -methyladenosine ( [Formula: see text]) plays an essential role in many vital biological processes. The identification of [Formula: see text] sites in RNAs is significantly important for both basic biomedical research and practical drug development. In this study, we designed a computational-based method, called TargetM6A, to rapidly and accurately target [Formula: see text] sites solely from the primary RNA sequences. Two new features, i.e., position-specific nucleotide/dinucleotide propensities (PSNP/PSDP), are introduced and combined with the traditional nucleotide composition (NC) feature to formulate RNA sequences. The extracted features are further optimized to obtain a much more compact and discriminative feature subset by applying an incremental feature selection (IFS) procedure. Based on the optimized feature subset, we trained TargetM6A on the training dataset with a support vector machine (SVM) as the prediction engine. We compared the proposed TargetM6A method with existing methods for predicting [Formula: see text] sites by performing stringent jackknife tests and independent validation tests on benchmark datasets. The experimental results show that the proposed TargetM6A method outperformed the existing methods for predicting [Formula: see text] sites and remarkably improved the prediction performances, with MCC = 0.526 and AUC = 0.818. We also provided a user-friendly web server for TargetM6A, which is publicly accessible for academic use at http://csbio.njust.edu.cn/bioinf/TargetM6A.

  1. Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

    PubMed

    Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

    2014-01-01

    Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

  2. Decaleside: a new class of natural insecticide targeting tarsal gustatory sites

    NASA Astrophysics Data System (ADS)

    Rajashekar, Yallappa; Rao, Lingamallu J. M.; Shivanandappa, Thimmappa

    2012-10-01

    Natural sources for novel insecticide molecules hold promise in view of their eco-friendly nature, selectivity, and mammalian safety. Recent progress in understanding the biology of insect olfaction and taste offers new strategies for developing selective pest control agents. We have isolated two natural insecticidal molecules from edible roots of Decalepis hamiltonii named Decalesides I and II, which are novel trisaccharides, highly toxic to household insect pests and stored-product insects. We have experimentally shown that insecticidal activity requires contact with tarsi on the legs but is not toxic orally. The insecticidal activity of molecules is lost by hydrolysis, and various sugars modify toxic response, showing that the insecticidal activity is via gustatory sites on the tarsi. Selective toxicity to insects by virtue of their gustatory site of action and the mammalian safety of the new insecticides is inherent in their chemical structure with 1-4 or 1-1 α linkage that is easily hydrolyzed by digestive enzymes of mammals. Decalesides represent a new chemical class of natural insecticides with a unique mode of action targeting tarsal chemosensory/gustatory system of insects.

  3. 75 FR 45158 - RBS Citizens, N.A., Business Services, Including On-Site Leased Workers of Manpower and Randstad...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... Citizens, N.A., Business Services, Including On-Site Leased Workers of Manpower and Randstad, 1 Citizens Drive, Riverside, RI RBS Citizens, N.A., Business Services, Including On-Site Leased Workers of Manpower... Worker Adjustment Assistance In accordance with Section 223 of the Trade Act of 1974, as amended (``Act...

  4. Novel GABA receptor pesticide targets.

    PubMed

    Casida, John E; Durkin, Kathleen A

    2015-06-01

    The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use

  5. 76 FR 81986 - Honeywell International, Inc., Automation and Control Solutions Division, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-29

    ..., Inc., Automation and Control Solutions Division, Including On-Site Leased Workers From Manpower... International, Inc., Automation and Control Solutions Division, Rock Island, Illinois. The notice was published...., Automation and Control Solutions Division. The Department has determined that these workers were sufficiently...

  6. 75 FR 41895 - Whirlpool Corporation, Evansville Division, Including On-Site Leased Workers From Andrews...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-19

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,585] Whirlpool Corporation, Evansville Division, Including On-Site Leased Workers From Andrews International, Inc., M.H. Equipment, and Kenco Logistics Services, LLC, Evansville, IN; Amended Certification Regarding Eligibility To Apply for...

  7. 77 FR 48550 - Technicolor Creative Services, Post Production Feature Mastering Division Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-14

    ... Services, Post Production Feature Mastering Division Including On-Site Leased Workers From Ajilon... Services, Post Production Feature Mastering Division, Hollywood, California (subject firm). The worker... the workers meet the eligibility requirements of the Trade Act of 1974. Conclusion After careful...

  8. Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala.

    PubMed

    Rizzo, Nidia; Gramajo, Rodrigo; Escobar, Maria Cabrera; Arana, Byron; Kroeger, Axel; Manrique-Saide, Pablo; Petzold, Max

    2012-10-30

    In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. The study was conducted as a cluster randomized community trial using "reduction of the vector population" as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical rains occurred in the area and

  9. Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

    PubMed Central

    2012-01-01

    Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical

  10. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

    PubMed

    Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

    2017-02-01

    We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

  11. Treating spondyloarthritis, including ankylosing spondylitis and psoriatic arthritis, to target: recommendations of an international task force

    PubMed Central

    Smolen, Josef S; Braun, Jürgen; Dougados, Maxime; Emery, Paul; FitzGerald, Oliver; Helliwell, Philip; Kavanaugh, Arthur; Kvien, Tore K; Landewé, Robert; Luger, Thomas; Mease, Philip; Olivieri, Ignazio; Reveille, John; Ritchlin, Christopher; Rudwaleit, Martin; Schoels, Monika; Sieper, Joachim; de Wit, Martinus; Baraliakos, Xenofon; Betteridge, Neil; Burgos-Vargas, Ruben; Collantes-Estevez, Eduardo; Deodhar, Atul; Elewaut, Dirk; Gossec, Laure; Jongkees, Merryn; Maccarone, Mara; Redlich, Kurt; van den Bosch, Filip; Wei, James Cheng-Chung; Winthrop, Kevin; van der Heijde, Désirée

    2014-01-01

    Background Therapeutic targets have been defined for diseases like diabetes, hypertension or rheumatoid arthritis and adhering to them has improved outcomes. Such targets are just emerging for spondyloarthritis (SpA). Objective To define the treatment target for SpA including ankylosing spondylitis and psoriatic arthritis (PsA) and develop recommendations for achieving the target, including a treat-to-target management strategy. Methods Based on results of a systematic literature review and expert opinion, a task force of expert physicians and patients developed recommendations which were broadly discussed and voted upon in a Delphi-like process. Level of evidence, grade and strength of the recommendations were derived by respective means. The commonalities between axial SpA, peripheral SpA and PsA were discussed in detail. Results Although the literature review did not reveal trials comparing a treat-to-target approach with another or no strategy, it provided indirect evidence regarding an optimised approach to therapy that facilitated the development of recommendations. The group agreed on 5 overarching principles and 11 recommendations; 9 of these recommendations related commonly to the whole spectrum of SpA and PsA, and only 2 were designed separately for axial SpA, peripheral SpA and PsA. The main treatment target, which should be based on a shared decision with the patient, was defined as remission, with the alternative target of low disease activity. Follow-up examinations at regular intervals that depend on the patient's status should safeguard the evolution of disease activity towards the targeted goal. Additional recommendations relate to extra-articular and extramusculoskeletal aspects and other important factors, such as comorbidity. While the level of evidence was generally quite low, the mean strength of recommendation was 9–10 (10: maximum agreement) for all recommendations. A research agenda was formulated. Conclusions The task force defined the

  12. The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

    PubMed

    Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

    2017-07-15

    Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

  13. Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

    PubMed

    Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

    2008-07-17

    Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

  14. α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

    PubMed Central

    Dong, Lan-Feng; Low, Pauline; Dyason, Jeffrey C.; Wang, Xiu-Fang; Prochazka, Lubomir; Witting, Paul K.; Freeman, Ruth; Swettenham, Emma; Valis, Karel; Liu, Ji; Zobalova, Renata; Turanek, Jaroslav; Spitz, Doug R.; Domann, Frederick E.; Scheffler, Immo E.; Ralph, Stephen J.; Neuzil, Jiri

    2009-01-01

    α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ) binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and undergo apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy. PMID:18372923

  15. Site Selection in Experiments: An Assessment of Site Recruitment and Generalizability in Two Scale-Up Studies

    ERIC Educational Resources Information Center

    Tipton, Elizabeth; Fellers, Lauren; Caverly, Sarah; Vaden-Kiernan, Michael; Borman, Geoffrey; Sullivan, Kate; Ruiz de Castilla, Veronica

    2016-01-01

    Recently, statisticians have begun developing methods to improve the generalizability of results from large-scale experiments in education. This work has included the development of methods for improved site selection when random sampling is infeasible, including the use of stratification and targeted recruitment strategies. This article provides…

  16. 78 FR 66780 - Blount International, Inc., Including On-Site Leased Workers From Express Employment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-06

    ... Express Employment Professionals, Portland, Oregon (TA-W-83,024) and Blount International, Carlton, Blount... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-83,024] Blount International, Inc., Including On-Site Leased Workers From Express Employment Professionals, Portland, Oregon [TA-W-83,024A...

  17. 75 FR 11914 - Chrysler, LLC, Mount Elliott Tool and Die, Including On-Site Leased Workers From Modern...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... Elliott Tool and Die, Including On-Site Leased Workers From Modern Professional Services, TAC Automotive... on-site leased workers from Modern Professional Services, TAC Automotive, Syncreon, CSC, and Resource... subject firm. The workers are engaged in the production of automotive dies and tooling. New information...

  18. 75 FR 28296 - Clark Engineering Co., Inc., Including On-Site Leased Workers From Kelly Services and Qualified...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-20

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,773] Clark Engineering Co... of Clark Engineering Co., Inc., including on-site leased workers of Kelly Services, Owosso, Michigan... from Qualified Staffing were employed on-site at the Owosso, Michigan location of Clark Engineering Co...

  19. 78 FR 48467 - Delphi Automotive Systems, LLC, Products and Service Solutions Division, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... amending this certification to include all workers of Delphi Automotive Systems, LLC, Product and Service..., LLC, Products and Service Solutions Division, Including On-Site Leased Workers From Bartech Workforce Management, Kokomo, Indiana; Amended Certification Regarding Eligibility To Apply for Worker Adjustment...

  20. 75 FR 52982 - Chrysler Group LLC, Formally Known as Chrysler LLC, Kenosha Engine Plant, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-30

    ... automobile engines. The company reports that workers leased from Syncreon were employed on-site at the..., Formally Known as Chrysler LLC, Kenosha Engine Plant, Including On-Site Leased Workers From Caravan Knight... Chrysler, LLC, Kenosha Engine Plant, Kenosha, Wisconsin. The notice was published in the Federal Register...

  1. 77 FR 3499 - FCI USA, LLC Corporate Administrative Division Including On-Site Leased Workers From JFC...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... Administrative Division Including On-Site Leased Workers From JFC Including Teleworkers Located Throughout the United States Reporting to Etters, PA; Amended Certification Regarding Eligibility To Apply for Worker.... 2273, the Department of Labor issued a Certification of Eligibility To Apply for Worker Adjustment...

  2. 76 FR 61740 - Geneon Entertainment (USA) Including On-Site Leased Workers From Interplace, Inc., Apple One and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-05

    ...) Including On-Site Leased Workers From Interplace, Inc., Apple One and Robert Half Legal Santa Monica, CA...-site leased workers from Interplace, Inc., and Apple One, Santa Monica, California. The workers are... from Interplace, Inc., Apple One and Robert Half Legal, Santa Monica, California, who became totally or...

  3. Targeted modulation of reactive oxygen species in the vascular endothelium.

    PubMed

    Shuvaev, Vladimir V; Muzykantov, Vladimir R

    2011-07-15

    'Endothelial cells lining vascular luminal surface represent an important site of signaling and injurious effects of reactive oxygen species (ROS) produced by other cells and endothelium itself in ischemia, inflammation and other pathological conditions. Targeted delivery of ROS modulating enzymes conjugated with antibodies to endothelial surface molecules (vascular immunotargeting) provides site-specific interventions in the endothelial ROS, unattainable by other formulations including PEG-modified enzymes. Targeting of ROS generating enzymes (e.g., glucose oxidase) provides ROS- and site-specific models of endothelial oxidative stress, whereas targeting of antioxidant enzymes SOD and catalase offers site-specific quenching of superoxide anion and H(2)O(2). These targeted antioxidant interventions help to clarify specific role of endothelial ROS in vascular and pulmonary pathologies and provide basis for design of targeted therapeutics for treatment of these pathologies. In particular, antibody/catalase conjugates alleviate acute lung ischemia/reperfusion injury, whereas antibody/SOD conjugates inhibit ROS-mediated vasoconstriction and inflammatory endothelial signaling. Encapsulation in protease-resistant, ROS-permeable carriers targeted to endothelium prolongs protective effects of antioxidant enzymes, further diversifying the means for targeted modulation of endothelial ROS. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. 75 FR 76037 - HAVI Logistics, North America a Subsidiary of HAVI Group, LP Including On-Site Leased Workers of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... Logistics, North America a Subsidiary of HAVI Group, LP Including On-Site Leased Workers of Express Personnel Services and the La Salle Network, Bloomingdale, IL; Havi Logistics, North America, Lisle, IL..., applicable to workers of HAVI Logistics, North America, a subsidiary of HAVI Group, LP, including on-site...

  5. Development of Bone Targeting Drugs.

    PubMed

    Stapleton, Molly; Sawamoto, Kazuki; Alméciga-Díaz, Carlos J; Mackenzie, William G; Mason, Robert W; Orii, Tadao; Tomatsu, Shunji

    2017-06-23

    The skeletal system, comprising bones, ligaments, cartilage and their connective tissues, is critical for the structure and support of the body. Diseases that affect the skeletal system can be difficult to treat, mainly because of the avascular cartilage region. Targeting drugs to the site of action can not only increase efficacy but also reduce toxicity. Bone-targeting drugs are designed with either of two general targeting moieties, aimed at the entire skeletal system or a specific cell type. Most bone-targeting drugs utilize an affinity to hydroxyapatite, a major component of the bone matrix that includes a high concentration of positively-charged Ca 2+ . The strategies for designing such targeting moieties can involve synthetic and/or biological components including negatively-charged amino acid peptides or bisphosphonates. Efficient delivery of bone-specific drugs provides significant impact in the treatment of skeletal related disorders including infectious diseases (osteoarthritis, osteomyelitis, etc.), osteoporosis, and metabolic skeletal dysplasia. Despite recent advances, however, both delivering the drug to its target without losing activity and avoiding adverse local effects remain a challenge. In this review, we investigate the current development of bone-targeting moieties, their efficacy and limitations, and discuss future directions for the development of these specific targeted treatments.

  6. Development of Bone Targeting Drugs

    PubMed Central

    Stapleton, Molly; Sawamoto, Kazuki; Alméciga-Díaz, Carlos J.; Mackenzie, William G.; Mason, Robert W.; Orii, Tadao; Tomatsu, Shunji

    2017-01-01

    The skeletal system, comprising bones, ligaments, cartilage and their connective tissues, is critical for the structure and support of the body. Diseases that affect the skeletal system can be difficult to treat, mainly because of the avascular cartilage region. Targeting drugs to the site of action can not only increase efficacy but also reduce toxicity. Bone-targeting drugs are designed with either of two general targeting moieties, aimed at the entire skeletal system or a specific cell type. Most bone-targeting drugs utilize an affinity to hydroxyapatite, a major component of the bone matrix that includes a high concentration of positively-charged Ca2+. The strategies for designing such targeting moieties can involve synthetic and/or biological components including negatively-charged amino acid peptides or bisphosphonates. Efficient delivery of bone-specific drugs provides significant impact in the treatment of skeletal related disorders including infectious diseases (osteoarthritis, osteomyelitis, etc.), osteoporosis, and metabolic skeletal dysplasia. Despite recent advances, however, both delivering the drug to its target without losing activity and avoiding adverse local effects remain a challenge. In this review, we investigate the current development of bone-targeting moieties, their efficacy and limitations, and discuss future directions for the development of these specific targeted treatments. PMID:28644392

  7. 76 FR 13227 - Continental Structural Plastics, Including On-Site Leased Workers From Kelly Services and Doepker...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-10

    ... Plastics, Including On-Site Leased Workers From Kelly Services and Doepker Group, Inc., Formerly Known As... Continental Structural Plastics, North Baltimore, Ohio. The workers produce exterior body panels and under... to TA-W-64,458 is hereby issued as follows: All workers of Continental Structural Plastics, including...

  8. Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae).

    PubMed

    Zimmer, Christoph T; Müller, Andreas; Heimbach, Udo; Nauen, Ralf

    2014-01-01

    Cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major pest of winter oilseed rape in several European countries particularly attacking young emerging plants in autumn. Over the last several decades, pyrethroid insecticides have been foliarly applied to control flea beetle outbreaks. Recent control failures in northern Germany suggested pyrethroid resistance development in cabbage stem flea beetles, which were confirmed by resistance monitoring bioassays using lambda-cyhalothrin in an adult vial test. The purpose of this study was to investigate the presence of polymorphisms in the para-type voltage-gated sodium channel gene of P. chrysocephala known to be involved in knock-down resistance (kdr). By using a degenerate primer approach we PCR amplified part of the para-type sodium channel gene and identified in resistant flea beetles a single nucleotide polymorphism resulting in an L1014F (kdr) mutation within domain IIS6 of the channel protein, known as one of the chief pyrethroid target-site resistance mechanisms in several other pest insects. Twenty populations including four archived museum samples collected between 1945 and 1958 were analyzed using a newly developed pyrosequencing diagnostic assay. The assay revealed a kdr allele frequency of 90-100% in those flea beetle populations expressing high-level cross-resistance in discriminating dose bioassays against different pyrethroids such as lambda-cyhalothrin, tau-fluvalinate, etofenprox and bifenthrin. The presence of target-site resistance to pyrethroids in cabbage stem flea beetle is extremely worrying considering the lack of effective alternative modes of action to control this pest in Germany and other European countries, and is likely to result in major control problems once it expands to other geographies. The striking fact that cabbage stem flea beetle is next to pollen beetle, Meligethes aeneus the second coleopteran pest in European winter oilseed rape resisting

  9. 78 FR 8587 - American Showa, Inc.; Blanchester Plant, Including On-Site Leased Workers From Adecco and Sims...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-06

    ....; Blanchester Plant, Including On-Site Leased Workers From Adecco and Sims Bros., Inc.; Blanchester, OH; Amended... workers from Sims Bros., Inc. were working on-site at the subject firm during the relevant period and that the services supplied by Sims Bros., Inc. were related to the production of gear boxes (and parts...

  10. Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

    PubMed

    Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

    2017-10-12

    Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

  11. 75 FR 69471 - Smurfit-Stone Container Corporation, Containerboard Mill, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-12

    ... Corporation, Containerboard Mill, Including On-Site Leased Workers From KMW Enterprises, Ontonagon, MI... to workers of Smurfit-Stone Container Corporation, Containerboard Mill, Ontonagon, Michigan. The... Ontonagon, Michigan location of Smurfit-Stone Container Corporation, Containerboard Mill. The Department has...

  12. 76 FR 2144 - Quest Diagnostics, Inc. Information Technology Help Desk Services Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-12

    .... Information Technology Help Desk Services Including On-Site Leased Workers From Modis, West Norriton, PA..., applicable to workers of Quest Diagnostics, Inc., Information Technology Help Desk Services, West Norriton... Quest Diagnostics, Inc., Information Technology Help Desk [[Page 2145

  13. Effectiveness of off-line and web-based promotion of health information web sites.

    PubMed

    Jones, Craig E; Pinnock, Carole B

    2002-01-01

    The relative effectiveness of off-line and web-based promotional activities in increasing the use of health information web sites by target audiences were compared. Visitor sessions were classified according to their method of arrival at the site (referral) as external web site, search engine, or "no referrer" (i.e., visitor arriving at the site by inputting URL or using bookmarks). The number of Australian visitor sessions correlated with no referrer referrals but not web site or search-engine referrals. Results showed that the targeted consumer group is more likely to access the web site as a result of off-line promotional activities. The properties of target audiences likely to influence the effectiveness of off-line versus on-line promotional strategies include the size of the Internet using population of the target audience, their proficiency in the use of the Internet, and the increase in effectiveness of off-line promotional activities when applied to locally defined target audiences.

  14. Evaluation of a novel virtual screening strategy using receptor decoy binding sites.

    PubMed

    Patel, Hershna; Kukol, Andreas

    2016-08-23

    Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated.

  15. 78 FR 48468 - M/A-Com Technology Solutions, Including On-Site Leased Workers of Kelly Temps and Aerotek CE...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ...-W-82,242 is hereby issued as follows: All workers of M/A-Com Technology Solutions, including on-site... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-80,242; TA-W-80,242A] M/A-Com.../A-Com Technology Solutions, Including On-Site Leased Workers of Kelly Temps and Aerotek CE, Long...

  16. 76 FR 13666 - Pitney Bowes, Inc., Mailing Solutions Management, Global Engineering Group, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-14

    ...., Mailing Solutions Management, Global Engineering Group, Including On-Site Leased Workers From Guidant... workers and former workers of Pitney Bowes, Inc., Mailing Solutions Management Division, Engineering... reviewed the certification to clarify the subject worker group's identity. Additional information revealed...

  17. The 50 Constellation Priority Sites

    NASA Technical Reports Server (NTRS)

    Noble, S.; Joosten, K.; Eppler, D.; Gruener, J.; Mendell, W.; French, R.; Plescia, J.; Spudis, P.; Wargo, M.; Robinson, M.; hide

    2009-01-01

    The Constellation program (CxP) has developed a list of 50 sites of interest on the Moon which will be targeted by the LRO narrow angle camera. The list has also been provided to the M team to supplement their targeting list. This list does not represent a "site selection" process; rather the goal was to find "representative" sites and terrains to understand the range of possible surface conditions for human lunar exploration to aid engineering design and operational planning. The list compilers leveraged heavily on past site selection work (e.g. Geoscience and a Lunar Base Workshop - 1988, Site Selection Strategy for a Lunar Outpost - 1990, Exploration Systems Architecture Study (ESAS) - 2005). Considerations included scientific, resource utilization, and operational merits, and a desire to span lunar terrain types. The targets have been organized into two "tiers" of 25 sites each to provide a relative priority ranking in the event of mutual interference. A LEAG SAT (special action team) was established to validate and recommend modifications to the list. This SAT was chaired by Dr. Paul Lucey. They provided their final results to CxP in May. Dr. Wendell Mendell will organize an on-going analysis of the data as they come down to ensure data quality and determine if and when a site has sufficient data to be retired from the list. The list was compiled using the best available data, however, it is understood that with the flood of new lunar data, minor modifications or adjustments may be required.

  18. [Comparative study between fast and slow induction of propofol given by target-controlled infusion: expected propofol concentration at the effect site. Randomized controlled trial].

    PubMed

    Simoni, Ricardo Francisco; Miziara, Luiz Eduardo de Paula Gomes; Esteves, Luis Otávio; Silva, Diógenes de Oliveira; Ribeiro, Cristina Alves; Smith, Mariana Oki; Paula, Leonardo Ferreira de; Cangiani, Luis Henrique

    2015-01-01

    studies have shown that rate of propofol infusion may influence the predicted propofol concentration at the effect site (Es). The aim of this study was to evaluate the Es predicted by the Marsh pharmacokinetic model (ke0 0.26min(-1)) in loss of consciousness during fast or slow induction. the study included 28 patients randomly divided into two equal groups. In slow induction group (S), target-controlled infusion (TCI) of propofol with plasma, Marsh pharmacokinetic model (ke0 0.26min(-1)) with target concentration (Tc) at 2.0-μg.mL(-1) were administered. When the predicted propofol concentration at the effect site (Es) reached half of Es value, Es was increased to previous Es + 1μg.mL(-1), successively, until loss of consciousness. In rapid induction group (R), patients were induced with TCI of propofol with plasma (6.0μg.ml(-1)) at Es, and waited until loss of consciousness. in rapid induction group, Tc for loss of consciousness was significantly lower compared to slow induction group (1.67±0.76 and 2.50±0.56μg.mL(-1), respectively, p=0.004). the predicted propofol concentration at the effect site for loss of consciousness is different for rapid induction and slow induction, even with the same pharmacokinetic model of propofol and the same balance constant between plasma and effect site. Copyright © 2014 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

  19. 77 FR 40637 - Smurfit-Stone Container Corporation, Containerboard Mill, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-10

    ... Corporation, Containerboard Mill, Including On-Site Leased Workers From KMW Enterprises and General Security... Assistance on May 6, 2010, applicable to workers of Smurfit-Stone Container Corporation, Containerboard Mill... Ontonagon, Michigan location of Smurfit-Stone Container Corporation, Containerboard Mill, Ontonagon...

  20. 77 FR 13352 - Exxonmobil Chemical Company Films Business Division Including on-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-06

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,325] Exxonmobil Chemical..., applicable to ExxonMobil Chemical Company, Films Business Division, including on-site leased workers from... Chemical Company, Films Business Division. The Department has determined that these workers were...

  1. 78 FR 1265 - Dana Holding Corporation; Power Technologies Group Division; Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-08

    ...; Power Technologies Group Division; Including On-Site Leased Workers From Manpower Milwaukee, WI; Notice... former workers of Dana Holding Company, Power Technologies Group Division, Milwaukee, Wisconsin (subject... reconsideration investigation, I determine that workers of Dana Holding Company, Power Technologies Group Division...

  2. ExoMars 2018: the four final candidate Landing Sites

    NASA Astrophysics Data System (ADS)

    Loizeau, Damien; Flahaut, Jessica; Vago, Jorge L.; Hauber, Ernst; Bridges, John C.

    2015-04-01

    The ExoMars 2018 mission will land a rover on Mars, its scientific objectives are to search for signs of past and present life on Mars and to investigate the water/geochemical environment as a function of depth in the shallow subsurface. The rover will be able to travel several kilometres, analyzing surface and subsurface samples, down to a 2 meter depth. The very powerful combination of mobility with the ability to access in-depth locations, where organic molecules can be well preserved, is unique to this mission [1]. An invitation has been sent to the community to propose scientifically compelling sites for the mission [2], which comply to the main engineering constraints for landing and operation safety. Scientifically interesting sites include locations with evidence for long duration or frequently recurring aqueous activity, low energy transport and deposition, fined-grained, recently exposed sediments, and/or hydrated minerals such as clays or evaporites. The outcrops of interest must be distributed over the landing ellipse to ensure that the rover can access some of them over a short distance [2]. The received proposals have been reviewed by the Landing Site Selection Working Group (LSSWG) and at first eight sites were found to be compliant with the science, engineering, and planetary protection requirements [3]. These sites were presented by their proposers and discussed at the first landing site workshop that took place in ESAC, Spain, 26-28 March 2014. Following that workshop, four sites were selected for further investigation, on the base of their higher potential for long lived water activity, the presence of fine grained sediments, and also importantly on the high concentration of potential targets of interest over the whole landing ellipse [3]. The analysis of these sites, both in term of scientific relevance and engineering safety, is still on-going. Latest findings were presented during a second workshop that took place in ALTEC, Torino, Italy, 11

  3. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

    PubMed

    Li, Jun; Meng, Xiangbing; Zong, Yuan; Chen, Kunling; Zhang, Huawei; Liu, Jinxing; Li, Jiayang; Gao, Caixia

    2016-09-12

    Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

  4. Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

    PubMed

    Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

    2014-12-05

    As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

  5. 77 FR 29363 - Chicopee Inc., a Subsidiary of Polymer Group, Inc., Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-17

    ... Subsidiary of Polymer Group, Inc., Including On- Site Leased Workers From Manpower Staffing, North Little..., applicable to workers and former workers of Chicopee, Inc., a subsidiary of Polymer Group, Inc., including on... hereby issued as follows: All workers from Chicopee, Inc., a subsidiary of Polymer Group, Inc., including...

  6. DeepMirTar: a deep-learning approach for predicting human miRNA targets.

    PubMed

    Wen, Ming; Cong, Peisheng; Zhang, Zhimin; Lu, Hongmei; Li, Tonghua

    2018-06-01

    MicroRNAs (miRNAs) are small noncoding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression by targeting messenger RNAs (mRNAs). Because the underlying mechanisms associated with miRNA binding to mRNA are not fully understood, a major challenge of miRNA studies involves the identification of miRNA-target sites on mRNA. In silico prediction of miRNA-target sites can expedite costly and time-consuming experimental work by providing the most promising miRNA-target-site candidates. In this study, we reported the design and implementation of DeepMirTar, a deep-learning-based approach for accurately predicting human miRNA targets at the site level. The predicted miRNA-target sites are those having canonical or non-canonical seed, and features, including high-level expert-designed, low-level expert-designed, and raw-data-level, were used to represent the miRNA-target site. Comparison with other state-of-the-art machine-learning methods and existing miRNA-target-prediction tools indicated that DeepMirTar improved overall predictive performance. DeepMirTar is freely available at https://github.com/Bjoux2/DeepMirTar_SdA. lith@tongji.edu.cn, hongmeilu@csu.edu.cn. Supplementary data are available at Bioinformatics online.

  7. 75 FR 63508 - Warner Chilcott Pharmaceuticals, Inc. Including On-Site Leased Workers From Adecco Engineering...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-15

    ... Pharmaceuticals, Inc. Including On-Site Leased Workers From Adecco Engineering and Technical, Norwich, New York; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with... a Certification of Eligibility to Apply for Worker Adjustment Assistance on September 10, 2010...

  8. 75 FR 11922 - Apria Healthcare, Including On-Site Leased Workers From Corestaff, Ultimate Staffing (Roth...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,054C] Apria Healthcare..., 2009, applicable to workers of Apria Healthcare, including on-site leased workers from Corestaff... subject firm. The workers are engaged in activities related to the information technology and patient...

  9. 78 FR 48467 - Wausau Paper, Brainerd Converting Operation, Including On-Site Leased Workers From Employment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... were sufficiently under the control of the subject firm to be considered leased workers. Based on these... Converting Operation, Including On-Site Leased Workers From Employment Resource Center, Securitas and Marsden, Brainerd, Minnesota; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance...

  10. 78 FR 52982 - Regal Beloit Corporation; Springfield, Missouri Division Including On-site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-27

    ... these workers were sufficiently under the control of the subject firm to be considered leased workers...; Springfield, Missouri Division Including On-site Leased Workers From Penmac Personnel Services and GCA Services Group Springfield, Missouri; Amended Certification Regarding Eligibility to Apply for Worker...

  11. Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

    PubMed

    Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

    2015-07-01

    Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments. © 2015 John Wiley & Sons Ltd.

  12. TargetSpy: a supervised machine learning approach for microRNA target prediction.

    PubMed

    Sturm, Martin; Hackenberg, Michael; Langenberger, David; Frishman, Dmitrij

    2010-05-28

    Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences.In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila

  13. TargetSpy: a supervised machine learning approach for microRNA target prediction

    PubMed Central

    2010-01-01

    Background Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. Results We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences. In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Conclusion Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well

  14. 76 FR 4724 - Polaris Industries, Including On-Site Leased Workers From Westaff and Supply Technologies...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-26

    .... The Department has determined that these workers were sufficiently under the control of Polaris..., Including On-Site Leased Workers From Westaff and Supply Technologies, Osceola, WI; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with section 223 of the Trade...

  15. 77 FR 51064 - Dana Holding Corporation, Power Technologies Group Division, Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-23

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,317] Dana Holding Corporation, Power Technologies Group Division, Including On-Site Leased Workers From Manpower, Milwaukee, WI; Notice... investigation resulted in a negative determination based on the findings that the subject firm did not shift...

  16. 76 FR 14101 - Meadwestvaco Corporation, Consumer and Office Products Division, Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-15

    ... undated planning and organizing products. The review shows that on August 21, 2008, a certification of..., Consumer and Office Products Division, Including On-Site Leased Workers From Pro-Tel People, Sidney, NY; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with...

  17. RNA interference as a method for target-site screening in the Western Corn Rootworm, Diabrotica virgifera virgifera

    USDA-ARS?s Scientific Manuscript database

    RNA interference (RNAi) is one of the most powerful and extraordinarily-specific means by which to silence genes. The ability of RNAi to silence genes makes it possible to ascertain function from genomic data, thereby making it an excellent choice for target-site screening. To test the efficacy of...

  18. 77 FR 53913 - River Bend Industries, LLC, Including On-Site Leased Workers From FirstStaff, Trac Staffing, and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-04

    ..., LLC, Including On-Site Leased Workers From FirstStaff, Trac Staffing, and Worksource, Inc., Fort Smith... Staffing, Worksource, Inc., Fort Smith, Arkansas. The Department's notice of determination was published in... Worksource, Inc. were employed on-site at the Fort Smith, Arkansas location of River Bend Industries, LLC...

  19. Glioma Dual-Targeting Nanohybrid Protein Toxin Constructed by Intein-Mediated Site-Specific Ligation for Multistage Booster Delivery

    PubMed Central

    Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Li, Dongdong; Xu, Fan; Wu, Aihua; Wang, Jinyu; Huang, Yongzhuo

    2017-01-01

    Malignant glioma is one of the most untreatable cancers because of the formidable blood-brain barrier (BBB), through which few therapeutics can penetrate and reach the tumors. Biologics have been booming in cancer therapy in the past two decades, but their application in brain tumor has long been ignored due to the impermeable nature of BBB against effective delivery of biologics. Indeed, it is a long unsolved problem for brain delivery of macromolecular drugs, which becomes the Holy Grail in medical and pharmaceutical sciences. Even assisting by targeting ligands, protein brain delivery still remains challenging because of the synthesis difficulties of ligand-modified proteins. Herein, we propose a rocket-like, multistage booster delivery system of a protein toxin, trichosanthin (TCS), for antiglioma treatment. TCS is a ribosome-inactivating protein with the potent activity against various solid tumors but lack of specific action and cell penetration ability. To overcome the challenge of its poor druggability and site-specific modification, intein-mediated ligation was applied, by which a gelatinase-cleavable peptide and cell-penetrating peptide (CPP)-fused recombinant TCS toxin can be site-specifically conjugated to lactoferrin (LF), thus constructing a BBB-penetrating, gelatinase-activatable cell-penetrating nanohybrid TCS toxin. This nanohybrid TCS system is featured by the multistage booster strategy for glioma dual-targeting delivery. First, LF can target to the BBB-overexpressing low-density lipoprotein receptor-related protein-1 (LRP-1), and assist with BBB penetration. Second, once reaching the tumor site, the gelatinase-cleavable peptide acts as a separator responsive to the glioma-associated matrix metalloproteinases (MMPs), thus releasing to the CPP-fused toxin. Third, CPP mediates intratumoral and intracellular penetration of TCS toxin, thereby enhancing its antitumor activity. The BBB penetration and MMP-2-activability of this delivery system were

  20. Site Targeted Press Coated Delivery of Methylprednisolone Using Eudragit RS 100 and Chitosan for Treatment of Colitis.

    PubMed

    Jagdale, Swati; Chandekar, Apoorva

    2016-01-01

    Inflammatory bowel disease (IBD) is one of the five most prevalent gastrointestinal disease burdens which commonly require lifetime care. Worldwide incidence rate of ulcerative colitis and Crohn's disease is about 16.8% and 13.4% respectively. Colitis is an inflammation of the colon. Colon targeted drug delivery will direct the drug to the colon. The drug will reach at the site of action and hence its side effects as well as dose can be reduced. Recent patent describes treatment of ulcerative colitis using anti CD3 antibodies, with nicotine and anti-depressant drugs, budesonide foam etc. Present study deals with optimization of site targeted methylprednisolone delivery for treatment of colitis. Chitosan and Eudragit RS 100 were used as coating polymers. Tablets were prepared by press coated technology. The core tablets contain drug, avicel as binder, croscarmellose sodium as super disintegrant and dicalcium phosphate as diluent. Drug excipient compatibility was carried out using FTIR, UV and DSC. Design of experiment was used to optimize the formulation. Tablets were evaluated for thickness, weight variation, hardness, swelling index, in-vitro drug release and release of drug in simulated media. Optimized batch (B2) contained chitosan 40% and eudragit RS 100 17.5%. B2 showed in-vitro drug release 85.65 ± 7.6% in 6.8 pH phosphate buffer and 96.7 ±9.1% in simulated media after 7.5 hours. In-vivo x-ray placebo study for formulation B2 had shown that the tablet reached to the ascending colon after 5 hours. This indicated a potential site targeted delivery of optimized batch B2.

  1. Quantum mechanical design of enzyme active sites.

    PubMed

    Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

    2008-02-01

    The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

  2. 76 FR 10394 - Ohio Decorative Products, Inc., Including On-Site Leased Workers From Custom Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-24

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,890] Ohio Decorative Products, Inc., Including On-Site Leased Workers From Custom Staffing, Spencerville, OH; Amended Certification... Act of 1974, as amended (``Act''), 19 U.S.C. 2273, the Department of Labor issued a Certification of...

  3. 75 FR 43565 - Johns Manville; Engineered Products Division, Including On-Site Leased Workers From Volt...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-26

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,494] Johns Manville; Engineered Products Division, Including On-Site Leased Workers From Volt Workforce Solutions; Spartanburg, SC; Notice of Revised Determination on Reconsideration By application dated May 2, 2010, a petitioner requested administrative reconsideration of the...

  4. 77 FR 41807 - New Gear Process, a Division of Magna Powertrain, Including On-Site Leased Workers From ABM...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-16

    ... Service Northeast, Inc. were employed on-site at the East Syracuse, New York location of New Process Gear... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,940] New Gear Process, a Division of Magna Powertrain, Including On- Site Leased Workers From ABM Janitorial Service Northeast, Inc...

  5. MAPK Target Sites of Eyes Absent Are Not Required for Eye Development or Survival in Drosophila

    PubMed Central

    Jusiak, Barbara; Abulimiti, Abuduaini; Haelterman, Nele; Chen, Rui; Mardon, Graeme

    2012-01-01

    Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that plays an essential role in eye development and survival in Drosophila. Ectopic eye induction assays using cDNA transgenes have suggested that mitogen activated protein kinase (MAPK) activates Eya by phosphorylating it on two consensus target sites, S402 and S407, and that this activation potentiates the ability of Eya to drive eye formation. However, this mechanism has never been tested in normal eye development. In the current study, we generated a series of genomic rescue transgenes to investigate how loss- and gain-of-function mutations at these two MAPK target sites within Eya affect Drosophila survival and normal eye formation: eya+GR, the wild-type control; eyaSAGR, which lacks phosphorylation at the two target residues; and eyaSDEGR, which contains phosphomimetic amino acids at the same two residues. Contrary to the previous studies in ectopic eye development, all eya genomic transgenes tested rescue both eye formation and survival equally effectively. We conclude that, in contrast to ectopic eye formation, MAPK-mediated phosphorylation of Eya on S402 and S407 does not play a role in normal development. This is the first study in Drosophila to evaluate the difference in outcomes between genomic rescue and ectopic cDNA-based overexpression of the same gene. These findings indicate similar genomic rescue strategies may prove useful for re-evaluating other long-standing Drosophila developmental models. PMID:23251383

  6. AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription.

    PubMed

    Takai, Hideki; Araki, Shouta; Mezawa, Masaru; Kim, Dong-Soon; Li, Xinyue; Yang, Li; Li, Zhengyang; Wang, Zhitao; Nakayama, Youhei; Ogata, Yorimasa

    2008-02-29

    Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

  7. 77 FR 6586 - GE Oil & Gas Operations, LLC Including On-Site Leased Workers From Adecco, Argus Technical, Inc...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-08

    ..., LLC Including On-Site Leased Workers From Adecco, Argus Technical, Inc., Fox Valley Metrology URS Corp... the certification for workers of the subject firm. New information shows that workers leased from Fox... from Fox Valley Metrology, URS Corp. and CompuCom working on-site at the Oshkosh, Wisconsin location of...

  8. 75 FR 49526 - Halliburton Company, Duncan Mfg., Including On-Site Leased Workers from Express Personnel...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-13

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-70,232; TA-W-70,232A] Halliburton Company, Duncan Mfg., Including On-Site Leased Workers from Express Personnel, Clayton Personnel Service, and Manpower Planning, Duncan, OK; Halliburton Company, Technology and Engineering Division, Finance and Administration Division, Duncan, OK;...

  9. 78 FR 8587 - Thomson Reuters, Finance Operations & Technology Division, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-06

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,755] Thomson Reuters, Finance Operations & Technology Division, Including On-Site Leased Workers From Adecco; Eagan, MN; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with Section 223 of the Trade Act of 1974, as amended (`...

  10. Design of ligand-targeted nanoparticles for enhanced cancer targeting

    NASA Astrophysics Data System (ADS)

    Stefanick, Jared F.

    Ligand-targeted nanoparticles are increasingly used as drug delivery vehicles for cancer therapy, yet have not consistently produced successful clinical outcomes. Although these inconsistencies may arise from differences in disease models and target receptors, nanoparticle design parameters can significantly influence therapeutic efficacy. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, this work evaluates the roles of polyethylene glycol (PEG) coating, ethylene glycol (EG) peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size on tumor targeting in a systematic manner. These parameters were analyzed in multiple disease models by targeting human epidermal growth factor receptor 2 (HER2) in breast cancer and very late antigen-4 (VLA-4) in multiple myeloma to demonstrate the widespread applicability of this approach. By increasing the hydrophilicity of the targeting peptide sequence and simultaneously optimizing the EG peptide-linker length, the in vitro cellular uptake of targeted liposomes was significantly enhanced. Specifically, including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake ~80-fold using an EG6 peptide-linker compared to ~10-fold using an EG45 linker. In vivo, targeted liposomes prepared in a traditional manner lacking the oligolysine chain demonstrated similar biodistribution and tumor uptake to non-targeted liposomes. However, by including the oligolysine chain, targeted liposomes using an EG45 linker significantly improved tumor uptake ~8-fold over non-targeted liposomes, while the use of an EG6 linker decreased tumor accumulation and uptake, owing to differences in cellular uptake kinetics, clearance mechanisms, and binding site barrier effects. To further improve tumor targeting and enhance the selectivity of targeted

  11. Binding-Site Assessment by Virtual Fragment Screening

    PubMed Central

    Huang, Niu; Jacobson, Matthew P.

    2010-01-01

    The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock ∼11000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors. PMID:20404926

  12. 76 FR 45879 - West, a Thomson Reuters Business, Thomson Reuters Legal, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-01

    ... Business, Thomson Reuters Legal, Including On-Site Leased Workers From Adecco, Including a Teleworker... for Worker Adjustment Assistance In accordance with section 223 of the Trade Act of 1974, as amended... Apply for Worker Adjustment Assistance on June 21, 2010, applicable to workers of West, A Thomson...

  13. 75 FR 11913 - Chrysler, LLC, Sterling Stamping Plant, Including On-Site Leased Workers from Caravan Knight...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... Stamping Plant, Including On-Site Leased Workers from Caravan Knight Facilities Management LLC, Sterling... stampings. New information shows that workers leased from Caravan Knight Facilities Management LLC were... workers leased from Caravan Knight Facilities [[Page 11914

  14. 75 FR 67770 - Sara Lee Corporation Including On-Site Leased Workers From EDS, Hewitt Packard, Sapphire...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-03

    ..., Deductions, Collections, Call Center, Information Technology, Accounts Payable, General Accounts, Financial Accounts, Payroll, and Employee Master Data Departments Including On-Site Leased Workers From ADECCO..., Deductions, Collections, Call Center, Information Technology, Accounts Payable, General Accounts, Financial...

  15. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site.

    PubMed

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S; Mkhize, Nonhlanhla N; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D; Labuschagne, Phillip; Louder, Mark K; Bailer, Robert T; Abdool Karim, Salim S; Mascola, John R; Williamson, Carolyn; Moore, Penny L; Kwong, Peter D; Morris, Lynn

    2016-11-15

    All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive

  16. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

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

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.

    ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report themore » isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to

  17. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    PubMed Central

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.

    2016-01-01

    ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site

  18. 77 FR 67399 - Hewlett-Packard Company, Design Delivery Organization (DDO), Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-09

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,739; TA-W-81,739A] Hewlett-Packard Company, Design Delivery Organization (DDO), Including On-Site Leased Workers From Manpower, Synova Inc., and Pinnacle Technical Resources, Corvallis, OR; Hewlett-Packard Company, Ink Jet & Web Services, World Wide Design Group, Vancouver, WA;...

  19. Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

    PubMed Central

    Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

    2014-01-01

    Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

  20. 75 FR 453 - FLSMidth, Inc., Cement Division, Product Engineering, Including On-Site Leased Workers of Aerotek...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-05

    ... Division, Product Engineering, Including On-Site Leased Workers of Aerotek Contract Engineering, Allied Personnel Services, Eastern Engineering, Hobbie Professional Services, Mccallion Staffing Specialists, Peak Technical Services, Inc., Yoh Engineering, and Clarke Consulting, Inc., Bethlehem, PA; Amended Certification...

  1. Integrin Targeted MR Imaging

    PubMed Central

    Tan, Mingqian; Lu, Zheng-Rong

    2011-01-01

    Magnetic resonance imaging (MRI) is a powerful medical diagnostic imaging modality for integrin targeted imaging, which uses the magnetic resonance of tissue water protons to display tissue anatomic structures with high spatial resolution. Contrast agents are often used in MRI to highlight specific regions of the body and make them easier to visualize. There are four main classes of MRI contrast agents based on their different contrast mechanisms, including T1, T2, chemical exchange saturation transfer (CEST) agents, and heteronuclear contrast agents. Integrins are an important family of heterodimeric transmembrane glycoproteins that function as mediators of cell-cell and cell-extracellular matrix interactions. The overexpressed integrins can be used as the molecular targets for designing suitable integrin targeted contrast agents for MR molecular imaging. Integrin targeted contrast agent includes a targeting agent specific to a target integrin, a paramagnetic agent and a linker connecting the targeting agent with the paramagnetic agent. Proper selection of targeting agents is critical for targeted MRI contrast agents to effectively bind to integrins for in vivo imaging. An ideal integrin targeted MR contrast agent should be non-toxic, provide strong contrast enhancement at the target sites and can be completely excreted from the body after MR imaging. An overview of integrin targeted MR contrast agents based on small molecular and macromolecular Gd(III) complexes, lipid nanoparticles and superparamagnetic nanoparticles is provided for MR molecular imaging. By using proper delivery systems for loading sufficient Gd(III) chelates or superparamagnetic nanoparticles, effective molecular imaging of integrins with MRI has been demonstrated in animal models. PMID:21547154

  2. 78 FR 22909 - Mondelez Global LLC, Business Services Center, Including On-Site Leased Workers From Abacus...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-17

    ... Solutions, American Cybersystems, Inc., Collabera, Hewlett-Packard, Kelly Services, Kforce, Inc., Lancesoft..., Business Services Center, including on-site leased workers from Abacus Service Corporation, American Cyber..., American CyberSystems, Inc., Collabera, Hewlett-Packard, Kelly Services, Kforce, Inc., Lancesoft...

  3. Selection of the Mars Science Laboratory landing site

    USGS Publications Warehouse

    Golombek, M.; Grant, J.; Kipp, D.; Vasavada, A.; Kirk, Randolph L.; Fergason, Robin L.; Bellutta, P.; Calef, F.; Larsen, K.; Katayama, Y.; Huertas, A.; Beyer, R.; Chen, A.; Parker, T.; Pollard, B.; Lee, S.; Hoover, R.; Sladek, H.; Grotzinger, J.; Welch, R.; Dobrea, E. Noe; Michalski, J.; Watkins, M.

    2012-01-01

    The selection of Gale crater as the Mars Science Laboratory landing site took over five years, involved broad participation of the science community via five open workshops, and narrowed an initial >50 sites (25 by 20 km) to four finalists (Eberswalde, Gale, Holden and Mawrth) based on science and safety. Engineering constraints important to the selection included: (1) latitude (±30°) for thermal management of the rover and instruments, (2) elevation (<-1 km) for sufficient atmosphere to slow the spacecraft, (3) relief of <100-130 m at baselines of 1-1000 m for control authority and sufficient fuel during powered descent, (4) slopes of <30° at baselines of 2-5 m for rover stability at touchdown, (5) moderate rock abundance to avoid impacting the belly pan during touchdown, and (6) a radar-reflective, load-bearing, and trafficable surface that is safe for landing and roving and not dominated by fine-grained dust. Science criteria important for the selection include the ability to assess past habitable environments, which include diversity, context, and biosignature (including organics) preservation. Sites were evaluated in detail using targeted data from instruments on all active orbiters, and especially Mars Reconnaissance Orbiter. All of the final four sites have layered sedimentary rocks with spectral evidence for phyllosilicates that clearly address the science objectives of the mission. Sophisticated entry, descent and landing simulations that include detailed information on all of the engineering constraints indicate all of the final four sites are safe for landing. Evaluation of the traversabilty of the landing sites and target “go to” areas outside of the ellipse using slope and material properties information indicates that all are trafficable and “go to” sites can be accessed within the lifetime of the mission. In the final selection, Gale crater was favored over Eberswalde based on its greater diversity and potential habitability.

  4. Ambient airborne solids concentrations including volcanic ash at Hanford, Washington sampling sites subsequent to the Mount St. Helens eruption

    NASA Technical Reports Server (NTRS)

    Sehmel, G. A.

    1982-01-01

    Airborne solids concentrations were measured on a near daily basis at two Hanford, Washington sites after the eruption of Mount St. Helens on May 18, 1980. These sites are about 211 km east of Mount St. Helens. Collected airborne solids included resuspended volcanic ash plus normal ambient solids. Average airborne solids concentrations were greater at the Hanford meteorological station sampling site which is 24 km northwest of the Horn Rapids dam sampling site. These increased concentrations reflect the sampling site proximity to greater ash fallout depths. Both sites are in low ash fallout areas although the Hanford meteorological station site is closer to the greater ash fallout areas. Airborne solids concentrations were decreased by rain, but airborne solids concentrations rapidly increased as surfaces dried. Airborne concentrations tended to become nearly the same at both sampling sites only for July 12 and 13.

  5. 75 FR 11920 - Agilent Technologies, Eesof Division, Including On-Site Leased Workers From Volt and Managed...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... software and related services including quality assurance and learning products, marketing, product development, marketing and administration. The company reports that on-site leased workers from Managed..., Santa Clara, California, and the Everett, Washington locations of Agilent Technologies, EEsof Division...

  6. Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

    PubMed Central

    Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

    2016-01-01

    Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

  7. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.

    PubMed

    Clop, Alex; Marcq, Fabienne; Takeda, Haruko; Pirottin, Dimitri; Tordoir, Xavier; Bibé, Bernard; Bouix, Jacques; Caiment, Florian; Elsen, Jean-Michel; Eychenne, Francis; Larzul, Catherine; Laville, Elisabeth; Meish, Françoise; Milenkovic, Dragan; Tobin, James; Charlier, Carole; Georges, Michel

    2006-07-01

    Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

  8. 75 FR 11920 - General Electric Lighting-Ravenna Lamp Plant, Lighting Division, Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,767] General Electric Lighting-Ravenna Lamp Plant, Lighting Division, Including On-Site Leased Workers from Devore Technologies, Ravenna..., 2009, applicable to workers of General Electric Lighting-Ravenna Lamp Plant, Lighting Division...

  9. 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)

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

    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) havemore » 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.« less

  10. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo.

    PubMed

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-01-01

    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro . Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo , we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

  11. Advances in targeted genome editing.

    PubMed

    Perez-Pinera, Pablo; Ousterout, David G; Gersbach, Charles A

    2012-08-01

    New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. 75 FR 43556 - Badger Meter, Inc., Including On-Site Leased Workers From Sourcepoint Staffing, Seek, and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-26

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,666] Badger Meter, Inc... of Badger Meter, Inc., including on-site leased workers from Sourcepoint Staffing, Milwaukee..., Wisconsin location of Badger Meter, Inc. The Department has determined that these workers were sufficiently...

  13. 75 FR 22628 - Cadence Innovation, LLC, Groesbeck Plant, Including On-Site Leased Workers from Michigan Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-29

    ... Human Capital Staffing (TA-W-64,715C); Cadence Innovation, LLC, Hillsdale Plant, Hillsdale, Michigan... Innovation, LLC, Groesbeck Plant, Including On-Site Leased Workers from Michigan Staffing, LLC, Modern Professional Services, LLC, TAC Transportation, Time Services, Inc., and Human Capital Staffing Clinton...

  14. 77 FR 19718 - Ford Motor Company Twin Cities Assembly Plant Vehicle Operations Division Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-02

    ... Cities Assembly Plant Vehicle Operations Division Including On-Site Leased Workers From AEROTEK, Albers... Industries, Waste Management, VMX, Nascote Industries, Delphi Electronics & Safety, Unicomm, And Pacer... Operations Division, St. Paul, Minnesota. The workers are engaged in activities related to the production of...

  15. 76 FR 2710 - Pitney Bowes, Inc., Mailing Solutions Management Division Including On-Site Leased Workers of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-14

    ...., Mailing Solutions Management Division Including On-Site Leased Workers of Guidant Group, and Teleworkers... Bowes, Inc., Mailing Solutions Management Division, Engineering Quality Assurance, Shelton, Connecticut... identity of the subject worker group. The worker group consists of workers of Pitney Bowes, Inc., the...

  16. 77 FR 43368 - Joerns Healthcare, LLC, Stevens Point, Wisconsin Division, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-24

    .... The Department has determined that these workers were sufficiently under the control of the subject..., Stevens Point, Wisconsin Division, Including On-Site Leased Workers From ABR, Aerotek, and Manpower, Stevens Point, WI; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance...

  17. 75 FR 879 - National Starch and Chemical Company Specialty Starches Division Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-06

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-62,282] National Starch and Chemical Company Specialty Starches Division Including On-Site Leased Workers From Page Employment, Island... Adjustment Assistance on December 13, 2007, applicable to workers of National Starch and Chemical Company...

  18. 76 FR 79221 - Android Industries Belvidere, LLC, Including On-Site Leased Workers From QPS Employment Group...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-21

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,072] Android Industries..., 2010, applicable to workers of Android Industries Belvidere, LLC, including on-site leased workers from... Belvidere, Illinois location of Android Industries Belvidere, LLC. The Department has determined that these...

  19. 76 FR 175 - Continental Structural Plastics Including On-Site Leased Workers From Kelly Services and Time...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-03

    ... Plastics Including On-Site Leased Workers From Kelly Services and Time Staffing; North Baltimore, OH... Adjustment Assistance on December 31, 2008, applicable to workers of Continental Structural Plastics, North... Baltimore, Ohio location of Continental Structural Plastics. The Department has determined that these...

  20. 75 FR 54388 - Rain Bird Corporation, Arizona Molding Division Including On-Site Leased Workers From Lumea...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-07

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,762] Rain Bird Corporation, Arizona Molding Division Including On-Site Leased Workers From Lumea Staffing Services, Tri-State Staffing Services and Remedy Staffing (AKA Select Staffing) Tucson, AZ; Amended Certification Regarding Eligibility...

  1. p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding.

    PubMed

    Itakura, Eisuke; Mizushima, Noboru

    2011-01-10

    Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

  2. Finding the target sites of RNA-binding proteins

    PubMed Central

    Li, Xiao; Kazan, Hilal; Lipshitz, Howard D; Morris, Quaid D

    2014-01-01

    RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201 PMID:24217996

  3. Recent Progress in the Design and Discovery of RXR Modulators Targeting Alternate Binding Sites of the Receptor.

    PubMed

    Su, Ying; Zeng, Zhiping; Chen, Ziwen; Xu, Dan; Zhang, Weidong; Zhang, Xiao-Kun

    2017-01-01

    Retinoid X receptors (RXRs) occupy a central position within the nuclear receptor superfamily. They not only function as important transcriptional factors but also exhibit diverse nongenomic biological activities. The pleiotropic actions of RXRs under both physiological and pathophysiological conditions confer RXRs important drug targets for the treatment of cancer, and metabolic and neurodegenerative diseases. RXR modulators have been studied for the purpose of developing both drug molecules and chemical tools for biological investigation of RXR. Development of RXR modulators has focused on small molecules targeting the canonical ligand-binding pocket. However, accumulating results have demonstrated that there are other binding mechanisms by which small molecules interact with RXR to act as RXR modulators. This review discusses the recent development in the design and discovery of RXR modulators with a focus on those targeting novel binding sites on RXR.

  4. Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein

    PubMed Central

    Kaever, Thomas; Meng, Xiangzhi; Matho, Michael H.; Schlossman, Andrew; Li, Sheng; Sela-Culang, Inbal; Ofran, Yanay; Buller, Mark; Crump, Ryan W.; Parker, Scott; Frazier, April; Crotty, Shane; Zajonc, Dirk M.; Peters, Bjoern

    2014-01-01

    ABSTRACT Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies. IMPORTANCE Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic

  5. 75 FR 26794 - International Paper Company Franklin Pulp & Paper Mill Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... Company Franklin Pulp & Paper Mill Including On-Site Leased Workers From Railserve, Franklin, VA; Amended... workers of International Paper Company, Franklin Pulp & Paper Mill, Franklin, Virginia. The notice was... Pulp & Paper Mill. The Department has determined that these workers were sufficiently under the control...

  6. 76 FR 27669 - Penske Logistics LLC, a Subsidiary of General Electric/Penske Corporation Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,897] Penske Logistics LLC, a Subsidiary of General Electric/Penske Corporation Including On-Site Leased Workers From Kelly Temporary... workers and former workers of Penske Logistics LLC, a subsidiary of General Electric/Penske Corporation...

  7. 77 FR 17525 - Cinram Distribution, LLC, a Subsidiary of Cinram International Income Fund, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-26

    ... International Income Fund had their wages reported through a separate unemployment insurance (UI) tax account..., a Subsidiary of Cinram International Income Fund, Including On-Site Leased Workers From Good People..., 2012, applicable to workers of Cinram Distribution, LLC, a subsidiary of Cinram International Income...

  8. 75 FR 30065 - Chrysler, LLC, Sterling Heights Assembly Plant Including On-Site Leased Workers From Caravan...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-28

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-65,762] Chrysler, LLC, Sterling Heights Assembly Plant Including On-Site Leased Workers From Caravan Knight Facilities Management LLC and... Chrysler, LLC, Sterling Heights Assembly Plant, Sterling Heights, Michigan. The notice was published in the...

  9. 75 FR 45160 - Ingersoll-Rand/Harrow Products, Inc., Formerly Known as Locknetics Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... account under the name Harrow Products, Inc. Accordingly, the Department is amending this certification to... Products, Inc., Formerly Known as Locknetics Including On-Site Leased Workers From Monroe Staffing Services, Adecco USA, Inc., and Infinistaff, LLC, Bristol, CT; Amended Certification Regarding Eligibility To Apply...

  10. 76 FR 30393 - Bush Industries, Inc., Including On-Site Leased Workers From Express Employment Professionals and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-25

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,995] Bush Industries, Inc... workers of Bush Industries, Inc., including on-site leased workers from Express Employment Professionals... wood furniture. The determination was issued on February 10, 2011. The Department's Notice will soon be...

  11. 76 FR 27365 - West, A Thomson Reuters Business, Thomson Reuters Legal Division, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-11

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-75,099] West, A Thomson Reuters Business, Thomson Reuters Legal Division, Including On-Site Leased Workers From ADECCO, Albuquerque, NM... Adjustment Assistance (TAA) applicable to workers and former workers of West, A Thomson Reuters Business...

  12. 78 FR 37586 - TE Connectivity, CIS-Appliances Division, Including On-Site Leased Workers From Kelly Services...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-21

    ... supply of administrative support services (in support of production). The worker group includes on-site... components produced by the subject workers. Further, aggregate imports of articles like or directly... reconsideration, the worker supplied new information regarding a possible shift in the production of like or...

  13. Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

    PubMed Central

    Mitchell, Sara N.; Rigden, Daniel J.; Dowd, Andrew J.; Lu, Fang; Wilding, Craig S.; Weetman, David; Dadzie, Samuel; Jenkins, Adam M.; Regna, Kimberly; Boko, Pelagie; Djogbenou, Luc; Muskavitch, Marc A. T.; Ranson, Hilary; Paine, Mark J. I.; Mayans, Olga; Donnelly, Martin J.

    2014-01-01

    The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae. PMID:24675797

  14. The Flavivirus Protease As a Target for Drug Discovery

    PubMed Central

    Brecher, Matthew; Zhang, Jing; Li, Hongmin

    2014-01-01

    Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures. PMID:24242363

  15. The flavivirus protease as a target for drug discovery.

    PubMed

    Brecher, Matthew; Zhang, Jing; Li, Hongmin

    2013-12-01

    Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.

  16. In silico target prediction for elucidating the mode of action of herbicides including prospective validation.

    PubMed

    Chiddarwar, Rucha K; Rohrer, Sebastian G; Wolf, Antje; Tresch, Stefan; Wollenhaupt, Sabrina; Bender, Andreas

    2017-01-01

    The rapid emergence of pesticide resistance has given rise to a demand for herbicides with new mode of action (MoA). In the agrochemical sector, with the availability of experimental high throughput screening (HTS) data, it is now possible to utilize in silico target prediction methods in the early discovery phase to suggest the MoA of a compound via data mining of bioactivity data. While having been established in the pharmaceutical context, in the agrochemical area this approach poses rather different challenges, as we have found in this work, partially due to different chemistry, but even more so due to different (usually smaller) amounts of data, and different ways of conducting HTS. With the aim to apply computational methods for facilitating herbicide target identification, 48,000 bioactivity data against 16 herbicide targets were processed to train Laplacian modified Naïve Bayesian (NB) classification models. The herbicide target prediction model ("HerbiMod") is an ensemble of 16 binary classification models which are evaluated by internal, external and prospective validation sets. In addition to the experimental inactives, 10,000 random agrochemical inactives were included in the training process, which showed to improve the overall balanced accuracy of our models up to 40%. For all the models, performance in terms of balanced accuracy of≥80% was achieved in five-fold cross validation. Ranking target predictions was addressed by means of z-scores which improved predictivity over using raw scores alone. An external testset of 247 compounds from ChEMBL and a prospective testset of 394 compounds from BASF SE tested against five well studied herbicide targets (ACC, ALS, HPPD, PDS and PROTOX) were used for further validation. Only 4% of the compounds in the external testset lied in the applicability domain and extrapolation (and correct prediction) was hence impossible, which on one hand was surprising, and on the other hand illustrated the utilization of

  17. Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin.

    PubMed

    Dostalova, Simona; Polanska, Hana; Svobodova, Marketa; Balvan, Jan; Krystofova, Olga; Haddad, Yazan; Krizkova, Sona; Masarik, Michal; Eckschlager, Tomas; Stiborova, Marie; Heger, Zbynek; Adam, Vojtech

    2018-06-11

    Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

  18. Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including protein markers, pathogens and cellular debris

    DOEpatents

    Martinez, Jennifer S [Santa Fe, NM; Swanson, Basil I [Los Alamos, NM; Grace, Karen M [Los Alamos, NM; Grace, Wynne K [Los Alamos, NM; Shreve, Andrew P [Santa Fe, NM

    2009-06-02

    An assay element is described including recognition ligands bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of a biological target is described including injecting a biological target-containing sample into a sensor cell including the assay element, with the recognition ligands adapted for binding to selected biological targets, maintaining the sample within the sensor cell for time sufficient for binding to occur between selected biological targets within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting the fluorescent-label in any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

  19. 77 FR 19719 - Whirlpool Corporation Including On-Site Leased Workers From Career Solutions TEC Staffing, IBM...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-02

    ... Including Workers Whose Unemployment Insurance (UI) Wages Are Reported Through U.S. Security Fort Smith, AR... Staffing, Fort Smith, Arkansas. The workers are engaged in the production of refrigerators and trash... information shows that workers leased from Andrews International employed on-site at the Fort Smith, Arkansas...

  20. 75 FR 11915 - Chrysler, LLC, Sterling Heights Vehicle Test Center, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-65,672] Chrysler, LLC, Sterling Heights Vehicle Test Center, Including On-Site Leased Workers From Caravan Knight Facilities Management LLC; Sterling Heights, MI; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance and Alternative Trade Adjustment...

  1. 76 FR 62451 - Avon Products, Inc., Including On-Site Leased Workers From Spherion/Source Right, Springdale...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-07

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,095] Avon Products, Inc., Including On-Site Leased Workers From Spherion/Source Right, Springdale, Ohio; Amended Certification... Act of 1974, as amended (``Act''), 19 U.S.C. 2273, the Department of Labor issued a Certification of...

  2. 75 FR 28297 - Ovonic Energy Products Including On-Site Leased Workers From PDSI Springboro, OH; Amended...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-20

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-70,055] Ovonic Energy Products Including On-Site Leased Workers From PDSI Springboro, OH; Amended Certification Regarding Eligibility To... amended (``Act''), 19 U.S.C. 2273, the Department of Labor issued a Certification of Eligibility to Apply...

  3. 78 FR 12358 - Cardinal Health, Financial Shared Services West, Including On-Site Leased Workers From Aerotek...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-22

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-82,253] Cardinal Health, Financial Shared Services West, Including On- Site Leased Workers From Aerotek, eXcel Staffing, Experis Finance (Manpower), Ricoh, USA, Dawson Creative, Mergis Group and Tailored Management, Albuquerque, NM; Amended Certification Regarding Eligibility To...

  4. 76 FR 29797 - International Automotive Components, North America, Including On-Site Leased Workers From At-Work...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-23

    ... Harvard Resources Solutions, Lebanon, VA; Amended Certification Regarding Eligibility To Apply for Worker..., including on-site leased workers from At- Work Personnel and CJR Solutions, d/b/a Harvard Resources... from At-Work Personnel and CJR Solutions, d/b/a Harvard Resource Solutions, Lebanon, Virginia, who...

  5. 75 FR 26791 - Chrysler, LLC, Trenton Engine Plant, Including On-Site Leased Workers from Caravan Knight...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-64,550] Chrysler, LLC, Trenton Engine Plant, Including On-Site Leased Workers from Caravan Knight Facilities Management LLC and Devon Facility Management, Trenton, MI, Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance and Alternative Trade...

  6. 75 FR 20389 - Findlay Industries, Inc., Findlay Ohio Plant One; Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-19

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-70,242] Findlay Industries, Inc., Findlay Ohio Plant One; Including On- Site Leased Workers From Alternative Management Resource, Inc. (AMRI of Findlay) Also Known as Alternative Management Temporary Services Findlay, OH; Amended Certification Regarding Eligibility To Apply for...

  7. Targeted enzyme prodrug therapies.

    PubMed

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  8. Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature

    PubMed Central

    Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Tang, Yisi; Wang, Huiyuan; Xu, Qin; Li, Yaping; Li, Feng; Huang, Yongzhuo

    2017-01-01

    Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation. PMID:27914267

  9. Increasing the Structural Coverage of Tuberculosis Drug Targets

    PubMed Central

    Baugh, Loren; Phan, Isabelle; Begley, Darren W.; Clifton, Matthew C.; Armour, Brianna; Dranow, David M.; Taylor, Brandy M.; Muruthi, Marvin M.; Abendroth, Jan; Fairman, James W.; Fox, David; Dieterich, Shellie H.; Staker, Bart L.; Gardberg, Anna S.; Choi, Ryan; Hewitt, Stephen N.; Napuli, Alberto J.; Myers, Janette; Barrett, Lynn K.; Zhang, Yang; Ferrell, Micah; Mundt, Elizabeth; Thompkins, Katie; Tran, Ngoc; Lyons-Abbott, Sally; Abramov, Ariel; Sekar, Aarthi; Serbzhinskiy, Dmitri; Lorimer, Don; Buchko, Garry W.; Stacy, Robin; Stewart, Lance J.; Edwards, Thomas E.; Van Voorhis, Wesley C.; Myler, Peter J.

    2015-01-01

    High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases. PMID:25613812

  10. Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.

    PubMed

    Steinbach, Denise; Gutbrod, Oliver; Lümmen, Peter; Matthiesen, Svend; Schorn, Corinna; Nauen, Ralf

    2015-08-01

    Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where

  11. Tibial anatomy in normal small breed dogs including anisometry of various extracapsular stabilizing suture attachment sites.

    PubMed

    Witte, P G

    2015-01-01

    To investigate proximal tibial anatomy and its influence on anisometry of extracapsular stabilizing sutures in small dog breeds. Mediolateral radiographs of the femora, stifles, and tibiae of 12 small breed dogs were acquired with the stifles positioned at various angles. Measurements taken included tibial plateau angle (TPA), diaphyseal: proximal tibial angle (DPA), patellar tendon angle (PTA), Z-angle, relative tibial tuberosity width (rTTW), and the distance between six combinations of two femoral and three tibial extra-capsular stabilizing suture (ECS) attachment sites. Theoretical strain through stifle range-of-motion was recorded. The TPA (32° ± 5.8°), DPA (10.2° ± 7.3°), PTA (103.7° ± 6.2°), and Z-angle (70.4° ± 9.0°) were positively correlated with one another (R >0.7), but none were correlated with rTTW (0.93 ± 0.10). The F2-T1 combination of ECS attachment sites had lowest strain for nine stifles. The shortest attachment site separation was at a stifle flexion of 50° for nine stifles. Proximal tibial anatomy measurements could not predict optimal attachment site combination, optimal stifle angle for suture placement, or ECS strain. There is individual variation in the optimal attachment site combination and stifle angle for suture placement, which may influence consistency of outcomes with ECS.

  12. The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

    PubMed

    Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

    2009-03-01

    The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

  13. Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

    PubMed Central

    Whisenant, Thomas C.; Ho, David T.; Benz, Ryan W.; Rogers, Jeffrey S.; Kaake, Robyn M.; Gordon, Elizabeth A.; Huang, Lan; Baldi, Pierre; Bardwell, Lee

    2010-01-01

    In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates. PMID:20865152

  14. 76 FR 16447 - Lafarge North America, Inc., a Subsidiary of Lafarge, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-23

    ... that workers leased from Industrial Services, Incorporated and Summit Building Maintenance were... leased workers from Industrial Services, Incorporated and Summit Building Maintenance, Seattle..., Inc., a Subsidiary of Lafarge, Including On-Site Leased Workers From Industrial Services, Incorporated...

  15. 76 FR 22729 - Chrysler Group, LLC, Power Train Division, Mack Avenue Engine Plant #1, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-22

    ..., Power Train Division, Mack Avenue Engine Plant 1, Including On-Site Leased Workers From Caravan Knight..., applicable to workers of Chrysler Group, LLC, Power Train Division, Mack Avenue Engine Plant 1, including on... all workers of Chrysler LLC, Mack Avenue Engine Plants 1 & 2, Power Train Division, Detroit, Michigan...

  16. 78 FR 66779 - CTS Automotive LLC, a Subsidiary of CTS Corporation Including On-Site Leased Workers From Metro...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-06

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-83,010] CTS Automotive LLC, a Subsidiary of CTS Corporation Including On- Site Leased Workers From Metro Staff and Aerotek Including Workers Whose Unemployment Insurance (UI) Wages Are Reported Through D&R Technology LLC, Carol Stream, Illinois; Amended Certification Regarding...

  17. 78 FR 61388 - TA-W-82,705, the Boeing Company Boeing Commercial Aircraft, (BCA) Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-03

    ..., (BCA) Including On-Site Leased Workers From Adecco, USA, Inc., Cascade Engineering, CDI Corporation...., Cascade Engineering, CDI Corporation, Chipton Ross, Inc., Comforce Corporation, Computer Staffing Services...

  18. p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

    PubMed Central

    Itakura, Eisuke

    2011-01-01

    Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway. PMID:21220506

  19. The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

    PubMed

    Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

    2018-05-04

    Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  20. Pediatric, Adolescent, and Young Adult Thyroid Carcinoma Harbors Frequent and Diverse Targetable Genomic Alterations, Including Kinase Fusions

    PubMed Central

    Schrock, Alexa B.; Anderson, Peter M.; Morris, John C.; Heilmann, Andreas M.; Holmes, Oliver; Wang, Kai; Johnson, Adrienne; Waguespack, Steven G.; Ou, Sai‐Hong Ignatius; Khan, Saad; Fung, Kar‐Ming; Stephens, Philip J.; Erlich, Rachel L.; Miller, Vincent A.; Ross, Jeffrey S.; Ali, Siraj M.

    2017-01-01

    Background. Thyroid carcinoma, which is rare in pediatric patients (age 0–18 years) but more common in adolescent and young adult (AYA) patients (age 15–39 years), carries the potential for morbidity and mortality. Methods. Hybrid‐capture‐based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). Results. GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety‐three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. Conclusion. CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy. Implications for Practice. The detection of diverse clinically relevant genomic alterations in the majority of pediatric, adolescent, and young adult patients with thyroid carcinoma in this study suggests that comprehensive genomic profiling may be beneficial for young

  1. Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

    PubMed

    Doolittle, Elizabeth; Peiris, Pubudu M; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P; Karathanasis, Efstathios

    2015-08-25

    Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

  2. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites

    PubMed Central

    Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

    2015-01-01

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. DOI: http://dx.doi.org/10.7554/eLife.11553.001 PMID:26609810

  3. Detection of canonical A-to-G editing events at 3' UTRs and microRNA target sites in human lungs using next-generation sequencing.

    PubMed

    Soundararajan, Ramani; Stearns, Timothy M; Griswold, Anthony L; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

    2015-11-03

    RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3' untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3' UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states.

  4. 76 FR 61742 - Unimin Corporation Including On-Site Leased Workers From Staffmark and Elwood Staffing Aurora, IN...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-05

    ... Unimin Corporation. The Department has determined that these workers were sufficiently under the control... Including On-Site Leased Workers From Staffmark and Elwood Staffing Aurora, IN; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance and Alternative Trade Adjustment Assistance...

  5. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    PubMed

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. Copyright © 2016. Published by Elsevier Ltd.

  6. Target-site resistance to neonicotinoids.

    PubMed

    Crossthwaite, Andrew J; Rendine, Stefano; Stenta, Marco; Slater, Russell

    2014-10-01

    Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

  7. Linking Oviposition Site Choice to Offspring Fitness in Aedes aegypti: Consequences for Targeted Larval Control of Dengue Vectors

    PubMed Central

    Wong, Jacklyn; Morrison, Amy C.; Stoddard, Steven T.; Astete, Helvio; Chu, Yui Yin; Baseer, Imaan; Scott, Thomas W.

    2012-01-01

    Background Current Aedes aegypti larval control methods are often insufficient for preventing dengue epidemics. To improve control efficiency and cost-effectiveness, some advocate eliminating or treating only highly productive containers. The population-level outcome of this strategy, however, will depend on details of Ae. aegypti oviposition behavior. Methodology/Principal Findings We simultaneously monitored female oviposition and juvenile development in 80 experimental containers located across 20 houses in Iquitos, Peru, to test the hypothesis that Ae. aegypti oviposit preferentially in sites with the greatest potential for maximizing offspring fitness. Females consistently laid more eggs in large vs. small containers (β = 9.18, p<0.001), and in unmanaged vs. manually filled containers (β = 5.33, p<0.001). Using microsatellites to track the development of immature Ae. aegypti, we found a negative correlation between oviposition preference and pupation probability (β = −3.37, p<0.001). Body size of emerging adults was also negatively associated with the preferred oviposition site characteristics of large size (females: β = −0.19, p<0.001; males: β = −0.11, p = 0.002) and non-management (females: β = −0.17, p<0.001; males: β = −0.11, p<0.001). Inside a semi-field enclosure, we simulated a container elimination campaign targeting the most productive oviposition sites. Compared to the two post-intervention trials, egg batches were more clumped during the first pre-intervention trial (β = −0.17, P<0.001), but not the second (β = 0.01, p = 0.900). Overall, when preferred containers were unavailable, the probability that any given container received eggs increased (β = 1.36, p<0.001). Conclusions/Significance Ae. aegypti oviposition site choice can contribute to population regulation by limiting the production and size of adults. Targeted larval control strategies may unintentionally lead to

  8. Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

    PubMed Central

    Fuller, James R; Rice, Phoebe A

    2017-01-01

    The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal. DOI: http://dx.doi.org/10.7554/eLife.21777.001 PMID:28177285

  9. Textpresso site-specific recombinases: A text-mining server for the recombinase literature including Cre mice and conditional alleles.

    PubMed

    Urbanski, William M; Condie, Brian G

    2009-12-01

    Textpresso Site Specific Recombinases (http://ssrc.genetics.uga.edu/) is a text-mining web server for searching a database of more than 9,000 full-text publications. The papers and abstracts in this database represent a wide range of topics related to site-specific recombinase (SSR) research tools. Included in the database are most of the papers that report the characterization or use of mouse strains that express Cre recombinase as well as papers that describe or analyze mouse lines that carry conditional (floxed) alleles or SSR-activated transgenes/knockins. The database also includes reports describing SSR-based cloning methods such as the Gateway or the Creator systems, papers reporting the development or use of SSR-based tools in systems such as Drosophila, bacteria, parasites, stem cells, yeast, plants, zebrafish, and Xenopus as well as publications that describe the biochemistry, genetics, or molecular structure of the SSRs themselves. Textpresso Site Specific Recombinases is the only comprehensive text-mining resource available for the literature describing the biology and technical applications of SSRs. (c) 2009 Wiley-Liss, Inc.

  10. Increasing the structural coverage of tuberculosis drug targets.

    PubMed

    Baugh, Loren; Phan, Isabelle; Begley, Darren W; Clifton, Matthew C; Armour, Brianna; Dranow, David M; Taylor, Brandy M; Muruthi, Marvin M; Abendroth, Jan; Fairman, James W; Fox, David; Dieterich, Shellie H; Staker, Bart L; Gardberg, Anna S; Choi, Ryan; Hewitt, Stephen N; Napuli, Alberto J; Myers, Janette; Barrett, Lynn K; Zhang, Yang; Ferrell, Micah; Mundt, Elizabeth; Thompkins, Katie; Tran, Ngoc; Lyons-Abbott, Sally; Abramov, Ariel; Sekar, Aarthi; Serbzhinskiy, Dmitri; Lorimer, Don; Buchko, Garry W; Stacy, Robin; Stewart, Lance J; Edwards, Thomas E; Van Voorhis, Wesley C; Myler, Peter J

    2015-03-01

    High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus "homolog-rescue" strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Increasing the structural coverage of tuberculosis drug targets

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

    Baugh, Loren; Phan, Isabelle; Begley, Darren W.

    High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. We found that of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structuresmore » would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PS APF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.« less

  12. Increasing the structural coverage of tuberculosis drug targets

    DOE PAGES

    Baugh, Loren; Phan, Isabelle; Begley, Darren W.; ...

    2014-12-19

    High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. We found that of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structuresmore » would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PS APF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.« less

  13. Comprehensive Calibration and Validation Site for Information Remote Sensing

    NASA Astrophysics Data System (ADS)

    Li, C. R.; Tang, L. L.; Ma, L. L.; Zhou, Y. S.; Gao, C. X.; Wang, N.; Li, X. H.; Wang, X. H.; Zhu, X. H.

    2015-04-01

    As a naturally part of information technology, Remote Sensing (RS) is strongly required to provide very precise and accurate information product to serve industry, academy and the public at this information economic era. To meet the needs of high quality RS product, building a fully functional and advanced calibration system, including measuring instruments, measuring approaches and target site become extremely important. Supported by MOST of China via national plan, great progress has been made to construct a comprehensive calibration and validation (Cal&Val) site, which integrates most functions of RS sensor aviation testing, EO satellite on-orbit caration and performance assessment and RS product validation at this site located in Baotou, 600km west of Beijing. The site is equipped with various artificial standard targets, including portable and permanent targets, which supports for long-term calibration and validation. A number of fine-designed ground measuring instruments and airborne standard sensors are developed for realizing high-accuracy stepwise validation, an approach in avoiding or reducing uncertainties caused from nonsynchronized measurement. As part of contribution to worldwide Cal&Val study coordinated by CEOS-WGCV, Baotou site is offering its support to Radiometric Calibration Network of Automated Instruments (RadCalNet), with an aim of providing demonstrated global standard automated radiometric calibration service in cooperation with ESA, NASA, CNES and NPL. Furthermore, several Cal&Val campaigns have been performed during the past years to calibrate and validate the spaceborne/airborne optical and SAR sensors, and the results of some typical demonstration are discussed in this study.

  14. 76 FR 81986 - Continuous Computing, Inc. (CCPU), A Subsidiary of RadiSys Corporation, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-29

    ..., Inc. (CCPU), A Subsidiary of RadiSys Corporation, Including On-Site Leased Workers From Qualstaff... Location, San Diego, CA; Amended Certification Regarding Eligibility To Apply for Worker Adjustment... Department of Labor issued a Certification of Eligibility to Apply for Worker Adjustment Assistance and...

  15. 75 FR 34170 - Chrysler Group LLC, Formally Known as Chrysler LLC, Kenosha Engine Plant, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ... automobile engines. The company reports that workers leased from Caravan Knight Facilities Management, LLC..., Formally Known as Chrysler LLC, Kenosha Engine Plant, Including On-Site Leased Workers From Caravan Knight..., Kenosha Engine Plant, Kenosha, Wisconsin. The notice was published in [[Page 34171

  16. 77 FR 29362 - Kohler Company, Malvern Division, Including On-Site Leased Workers From Manpower Staffing and Dow...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-17

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,299] Kohler Company, Malvern Division, Including On-Site Leased Workers From Manpower Staffing and Dow Cleaning Services, Malvern, AR; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with...

  17. 75 FR 38127 - Visteon Systems, LLC North Penn Plant Electronics Products Group Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-01

    ..., North Penn Plant, Electronics Products Group to be covered by this certification. The intent of the... North Penn Plant Electronics Products Group Including On-Site Leased Workers From Ryder Integrated... Certification Regarding Eligibility To Apply for Worker Adjustment Assistance and Alternative Trade Adjustment...

  18. Accident hazard evaluation and control decisions on forested recreation sites

    Treesearch

    Lee A. Paine

    1971-01-01

    Accident hazard associated with trees on recreation sites is inherently concerned with probabilities. The major factors include the probabilities of mechanical failure and of target impact if failure occurs, the damage potential of the failure, and the target value. Hazard may be evaluated as the product of these factors; i.e., expected loss during the current...

  19. Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo.

    PubMed

    Jedidi, Iness; Zhang, Fan; Qiu, Hongfang; Stahl, Stephen J; Palmer, Ira; Kaufman, Joshua D; Nadaud, Philippe S; Mukherjee, Sujoy; Wingfield, Paul T; Jaroniec, Christopher P; Hinnebusch, Alan G

    2010-01-22

    Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

  20. Thiamethoxam acts as a target-site synergist of spinosad in resistant strains of Frankliniella occidentalis.

    PubMed

    Guillén, Juan; Bielza, Pablo

    2013-02-01

    Previous studies have suggested that the resistance mechanism towards spinosad in Frankliniella occidentalis (Pergande) is an altered target site. Like the neonicotinoids, the spinosyns act on nicotinic acetylcholine receptors (nAChRs) in insects, but at a distinct site. The changes in nAChRs related to spinosad resistance in thrips might involve interaction with neonicotinoids. In this study, the efficacy of spinosad and neonicotinoids, alone and in combination, was evaluated in susceptible and spinosad-resistant thrips strains. The neonicotinoids tested were imidacloprid, thiacloprid, acetamiprid, thiamethoxam and clothianidin. No cross-resistance was shown between spinosad and any of the neonicotinoids. However, an increased toxicity was observed when a mixture of spinosad with thiamethoxam or clothianidin was tested. No synergism was found in the susceptible strains. The more spinosad-resistant the thrips strain, the stronger was the synergism. Data suggest that spinosad and thiamethoxam may interact at the nAChRs in spinosad-resistant thrips, facilitating enhanced insecticidal action. Copyright © 2012 Society of Chemical Industry.

  1. 77 FR 44677 - Quad/Graphics Inc., Including On-Site Leased Workers From Staff Mart and A.I.D., Jonesboro, AR...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-30

    ....,Including On-Site Leased Workers From Staff Mart and A.I.D.,Jonesboro, AR; Amended Certification Regarding... Inc., Jonesboro, Arkansas. The Department's notice of determination was published in the Federal... workers leased from Staff Mart and A.I.D. were employed on-site at the Jonesboro, Arkansas location of...

  2. Computational Tools for Allosteric Drug Discovery: Site Identification and Focus Library Design.

    PubMed

    Huang, Wenkang; Nussinov, Ruth; Zhang, Jian

    2017-01-01

    Allostery is an intrinsic phenomenon of biological macromolecules involving regulation and/or signal transduction induced by a ligand binding to an allosteric site distinct from a molecule's active site. Allosteric drugs are currently receiving increased attention in drug discovery because drugs that target allosteric sites can provide important advantages over the corresponding orthosteric drugs including specific subtype selectivity within receptor families. Consequently, targeting allosteric sites, instead of orthosteric sites, can reduce drug-related side effects and toxicity. On the down side, allosteric drug discovery can be more challenging than traditional orthosteric drug discovery due to difficulties associated with determining the locations of allosteric sites and designing drugs based on these sites and the need for the allosteric effects to propagate through the structure, reach the ligand binding site and elicit a conformational change. In this study, we present computational tools ranging from the identification of potential allosteric sites to the design of "allosteric-like" modulator libraries. These tools may be particularly useful for allosteric drug discovery.

  3. Moving target feature phenomenology data collection at China Lake

    NASA Astrophysics Data System (ADS)

    Gross, David C.; Hill, Jeff; Schmitz, James L.

    2002-08-01

    This paper describes the DARPA Moving Target Feature Phenomenology (MTFP) data collection conducted at the China Lake Naval Weapons Center's Junction Ranch in July 2001. The collection featured both X-band and Ku-band radars positioned on top of Junction Ranch's Parrot Peak. The test included seven targets used in eleven configurations with vehicle motion consisting of circular, straight-line, and 90-degree turning motion. Data was collected at 10-degree and 17-degree depression angles. Key parameters in the collection were polarization, vehicle speed, and road roughness. The collection also included a canonical target positioned at Junction Ranch's tilt-deck turntable. The canonical target included rotating wheels (military truck tire and civilian pick-up truck tire) and a flat plate with variable positioned corner reflectors. The canonical target was also used to simulate a rotating antenna and a vibrating plate. The target vehicles were instrumented with ARDS pods for differential GPS and roll, pitch and yaw measurements. Target motion was also documented using a video camera slaved to the X-band radar antenna and by a video camera operated near the target site.

  4. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  5. psRNATarget: a plant small RNA target analysis server

    PubMed Central

    Dai, Xinbin; Zhao, Patrick Xuechun

    2011-01-01

    Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/. PMID:21622958

  6. Measurement Sets and Sites Commonly Used for Characterization

    NASA Technical Reports Server (NTRS)

    Pagnutti, Mary; Holekamp, Kara; Ryan, Robert; Sellers, Richard; Davis, Bruce; Zanoni, Vicki

    2002-01-01

    Scientists at NASA's Earth Science Applications Directorate are creating a well-characterized Verification & Validation (V&V) site at the Stennis Space Center. This site enables the in-flight characterization of remote sensing systems and the data they acquire. The data are predominantly acquired by commercial, high spatial resolution satellite systems, such as IKONOS and QuickBird 2, and airborne systems. The smaller scale of these newer high resolution remote sensing systems allows scientists to characterize the geometric, spatial, and radiometric data properties using a single V&V site. The targets and techniques used to characterize data from these newer systems can differ significantly from the techniques used to characterize data from the earlier, coarser spatial resolution systems. Scientists are also using the SSC V&V site to characterize thermal infrared systems and active LIDAR systems. SSC employs geodetic targets, edge targets, radiometric tarps, and thermal calibration ponds to characterize remote sensing data products. This paper presents a proposed set of required measurements for visible through long-wave infrared remote sensing systems and a description of the Stennis characterization. Other topics discussed include: 1) The use of ancillary atmospheric and solar measurements taken at SSC that support various characterizations; 2) Additional sites used for radiometric, geometric, and spatial characterization in the continental United States; 3) The need for a standardized technique to be adopted by CEOS and other organizations.

  7. 76 FR 17447 - Penske Logistics LLC a Subsidiary of General Electric/Penske Corporation Including On-Site Leased...

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    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,897] Penske Logistics LLC a Subsidiary of General Electric/Penske Corporation Including On-Site Leased Workers From Kelly Temporary... (TAA) applicable to workers and former workers of Penske Logistics LLC, a subsidiary of General...

  8. 78 FR 52975 - Belden, Inc. Including On-Site Leased Workers From Adecco Horseheads, New York; Notice of...

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    2013-08-27

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-82,663] Belden, Inc. Including On-Site Leased Workers From Adecco Horseheads, New York; Notice of Affirmative Determination Regarding... review of the application, I conclude that the claim is of sufficient weight to justify reconsideration...

  9. 75 FR 55613 - Dupont Teijin Films Including On-Site Leased Workers From Schenkers Logistics, Inc., Florence, SC...

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    2010-09-13

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,259] Dupont Teijin Films... Teijin Films, including on-site leased workers from Schenkers Logistics, Inc., Florence, South Carolina... 59255). Workers are engaged in employment related to the production of polyester (PET) film. On our own...

  10. 76 FR 62452 - Avon Products, Inc. Including On-Site Leased Workers From Spherion/Source Right, Springdale, OH...

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    2011-10-07

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,095] Avon Products, Inc. Including On-Site Leased Workers From Spherion/Source Right, Springdale, OH; Amended Certification Regarding..., as amended (``Act''), 19 U.S.C. 2273, the Department of Labor issued a Certification of Eligibility...

  11. 78 FR 31593 - Aleris Recycling Bens Run, LLC, Including On-Site Leased Workers From Winans Extras Support...

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    2013-05-24

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-82,388] Aleris Recycling Bens Run, LLC, Including On-Site Leased Workers From Winans Extras Support Staffing and CDI Corporation... (TAA) applicable to workers and former workers of Aleris Recycling Bens Run, LLC, Friendly, West...

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

    PubMed Central

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

    2015-01-01

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

  13. Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

    PubMed

    Moretti, Marcelo L; Van Horn, Christopher R; Robertson, Renae; Segobye, Kabelo; Weller, Stephen C; Young, Bryan G; Johnson, William G; Douglas Sammons, R; Wang, Dafu; Ge, Xia; d' Avignon, André; Gaines, Todd A; Westra, Philip; Green, Amanda C; Jeffery, Taylor; Lespérance, Mackenzie A; Tardif, François J; Sikkema, Peter H; Christopher Hall, J; McLean, Michael D; Lawton, Mark B; Schulz, Burkhard

    2018-05-01

    The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. Target sites for chemical regulation of strigolactone signaling

    PubMed Central

    Nakamura, Hidemitsu; Asami, Tadao

    2014-01-01

    Demands for plant growth regulators (PGRs; chemicals that control plant growth) are increasing globally, especially in developing countries. Both positive and negative PGRs are widely used to enhance crop production and to suppress unwanted shoot growth, respectively. Strigolactones (SLs) are multifunctional molecules that function as phytohormones, inhibiting shoot branching and also functioning in the rhizospheric communication with symbiotic fungi and parasitic weeds. Therefore, it is anticipated that chemicals that regulate the functions of SLs will be widely used in agricultural applications. Although the SL biosynthetic pathway is not fully understood, it has been demonstrated that β-carotene isomerases, carotenoid cleavage dioxygenases (CCDs), and a cytochrome P450 monooxygenase are involved in strigolactone biosynthesis. A CCD inhibitor, abamine, which is also an inhibitor of abscisic acid biosynthesis, reduces the levels of SL in several plant species and reduces the germination rate of Orobanche minor seeds grown with tobacco. On the basis of the structure of abamine, several chemicals have been designed to specifically inhibit CCDs during SL synthesis. Cytochrome P450 monooxygenase is another target enzyme in the development of SL biosynthesis inhibitors, and the triazole-derived TIS series of chemicals is known to include SL biosynthesis inhibitors, although their target enzyme has not been identified. Recently, DWARF14 (D14) has been shown to be a receptor for SLs, and the D-ring moiety of SL is essential for its recognition by D14. A variety of SL agonists are currently under development and most agonists commonly contain the D-ring or a D-ring-like moiety. Several research groups have also resolved the crystal structure of D14 in the last two years. It is expected that this information on the D14 structure will be invaluable not only for developing SL agonists with novel structures but also in the design of inhibitors of SL receptors. PMID:25414720

  15. Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

    PubMed

    Luo, Ming; Gilbert, Brian; Ayliffe, Michael

    2016-07-01

    Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

  16. 76 FR 13667 - Chrysler Group LLC; Formerly Known as Chrysler LLC; Kenosha Engine Plant; Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-14

    ...; Formerly Known as Chrysler LLC; Kenosha Engine Plant; Including On-Site Leased Workers From Caravan Knight..., K+S Services, Inc., G4S Secure Solutions, Crassociates, Inc., CES, INC., Evans Distribution Systems... of Chrysler Group, LLC, formerly known as Chrysler, LLC, Kenosha Engine Plant, Kenosha, Wisconsin...

  17. 77 FR 3500 - Reading Powder Coatings, Inc., Including On-Site Leased Workers From Berks and Beyond Employment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-80,473] Reading Powder Coatings, Inc., Including On-Site Leased Workers From Berks and Beyond Employment Services and Gage Personnel Reading, PA; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance and Alternative Trade Adjustment Assistance In...

  18. 77 FR 14832 - RR Donnelley, Inc., Including On-Site Leased Workers From Manpower and Kelly Services, Bloomsburg...

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    2012-03-13

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-80,485] RR Donnelley, Inc., Including On-Site Leased Workers From Manpower and Kelly Services, Bloomsburg, PA; Notice of Affirmative... the claim is of sufficient weight to justify reconsideration of the U.S. Department of Labor's prior...

  19. 75 FR 26797 - Cummins Power Generation, Including On-Site Leased Workers of Adecco USA, Inc., Aerotek, Inc...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ..., Including On-Site Leased Workers of Adecco USA, Inc., Aerotek, Inc., the Bartech Group, Back Diamonds Networks, Entegee, Inc., DBA Midstates Technical, Manpower, Inc., Robert Half International, Summit... part that was used by a firm that employed a worker group currently eligible to apply for TAA. The...

  20. 75 FR 11918 - General Electric Kentucky Glass Plant, Lighting, LLC, Including On-Site Leased Workers From the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,011] General Electric Kentucky Glass Plant, Lighting, LLC, Including On-Site Leased Workers From the Patty Tipton Company, Aetna Building Maintenance, and Concentra, Lexington, KY; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordanc...

  1. 78 FR 47779 - Rough & Ready Lumber, LLC; Including On-Site Leased Workers From Perpetua Forests Company Cave...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-06

    ... Company were sufficiently under the control of the subject firm to be considered leased workers. Based on...; Including On-Site Leased Workers From Perpetua Forests Company Cave Junction, Oregon; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with Section 223 of the Trade...

  2. 77 FR 61028 - Hasbro, Inc.; Hasbro Managerial Services, Inc., Including On-Site Leased Workers of Entegee East...

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    2012-10-05

    ... relevant period and that the subject firm had sufficient control over the leased workers for the Department to determine that there was operational control of the leased workers by the subject firm. The... Managerial Services, Inc., Including On-Site Leased Workers of Entegee East Longmeadow, MA Amended...

  3. 77 FR 40636 - Northwest Hardwoods, Inc., Including On-Site Leased Workers of Manpower, Tacoma, WA; Notice of...

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    2012-07-10

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,314] Northwest Hardwoods, Inc., Including On-Site Leased Workers of Manpower, Tacoma, WA; Notice of Affirmative Determination Regarding... resulted in a negative determination based on the findings that there was no shift in production and there...

  4. 78 FR 74163 - Johnstown Specialty Castings Inc., a Subsidiary of WHEMCO, Including On-Site Leased Workers From...

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    2013-12-10

    ... Castings Inc., a Subsidiary of WHEMCO, Including On-Site Leased Workers From Berkebile Excavating Company... Assistance on June 25, 2013, applicable to workers of Johnstown Specialty Castings, Inc., a subsidiary of... Castings, Inc., a subsidiary of WHEMCO. The Department has determined that these workers were sufficiently...

  5. 77 FR 31643 - AI-Shreveport, LLC A Subsidiary of Android Industries Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-29

    ... Subsidiary of Android Industries Including On-Site Leased Workers From Career Adventures, Inc. Shreveport..., 2011, applicable to workers of AI-Shreveport, LLC, a subsidiary of Android Industries, Shreveport...- Shreveport, LLC, a subsidiary of Android Industries. The amended notice applicable to TA-W-80,515 is hereby...

  6. Structural basis for microRNA targeting

    DOE PAGES

    Schirle, Nicole T.; Sheu-Gruttadauria, Jessica; MacRae, Ian J.

    2014-10-31

    MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. In this paper, we determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing. Pairing to nt 2 to 5 promotes conformational changes that expose nt 2 to 8 and 13 to 16 for further target recognition. Interactions withmore » the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, whereas an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. Finally, these results explain the conserved nucleotide-pairing patterns in animal miRNA target sites first observed over two decades ago.« less

  7. Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster

    PubMed Central

    Remnant, Emily J.; Good, Robert T.; Schmidt, Joshua M.; Lumb, Christopher; Robin, Charles; Daborn, Phillip J.; Batterham, Philip

    2013-01-01

    The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 change into an Ala301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene. PMID:23959864

  8. Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins

    PubMed Central

    Kfoury, N.; Kapatos, G.

    2009-01-01

    CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain. PMID:19103292

  9. The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

    PubMed Central

    Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

    2009-01-01

    The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

  10. Chemical structure determines target organ carcinogenesis in rats

    PubMed Central

    Carrasquer, C. A.; Malik, N.; States, G.; Qamar, S.; Cunningham, S.L.; Cunningham, A.R.

    2012-01-01

    SAR models were developed for 12 rat tumour sites using data derived from the Carcinogenic Potency Database. Essentially, the models fall into two categories: Target Site Carcinogen – Non-Carcinogen (TSC-NC) and Target Site Carcinogen – Non-Target Site Carcinogen (TSC-NTSC). The TSC-NC models were composed of active chemicals that were carcinogenic to a specific target site and inactive ones that were whole animal non-carcinogens. On the other hand, the TSC-NTSC models used an inactive category also composed of carcinogens but to any/all other sites but the target site. Leave one out validations produced an overall average concordance value for all 12 models of 0.77 for the TSC-NC models and 0.73 for the TSC-NTSC models. Overall, these findings suggest that while the TSC-NC models are able to distinguish between carcinogens and non-carcinogens, the TSC-NTSC models are identifying structural attributes that associate carcinogens to specific tumour sites. Since the TSC-NTSC models are composed of active and inactive compounds that are genotoxic and non-genotoxic carcinogens, the TSC-NTSC models may be capable of deciphering non-genotoxic mechanisms of carcinogenesis. Together, models of this type may also prove useful in anticancer drug development since they essentially contain chemicals moieties that target specific tumour site. PMID:23066888

  11. Detection of canonical A-to-G editing events at 3′ UTRs and microRNA target sites in human lungs using next-generation sequencing

    PubMed Central

    Soundararajan, Ramani; Stearns, Timothy M.; Griswold, Anthony J.; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F.; King, Benjamin L.; Kolliputi, Narasaiah

    2015-01-01

    RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3′ untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3′ UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states. PMID:26486088

  12. 76 FR 2145 - Masco Builder Cabinet Group Including On-Site Leased Workers From Reserves Network, Jackson, OH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-12

    ...,287B; TA-W-71,287C] Masco Builder Cabinet Group Including On-Site Leased Workers From Reserves Network, Jackson, OH; Masco Builder Cabinet Group, Waverly, OH; Masco Builder Cabinet Group, Seal Township, OH; Masco Builder Cabinet Group, Seaman, OH; Amended Certification Regarding Eligibility To Apply for Worker...

  13. 75 FR 45166 - Jeld-Wen, Inc., Hawkins Window Division, Including On-Site Leased Workers of Nicolet Staffing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... Window Division, Including On-Site Leased Workers of Nicolet Staffing, Hawkins, WI; Notice of Negative... imports of wood- and aluminum- clad windows and patio doors did not contribute to worker group separations... of wood- and aluminum-clad windows and patio doors during 2007, 2008, January through April 2008, and...

  14. Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis

    PubMed Central

    Doolittle, Elizabeth; Peiris, Pubudu M.; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P.; Karathanasis, Efstathios

    2015-01-01

    Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection. PMID:26203676

  15. Membrane Transporters: Structure, Function and Targets for Drug Design

    NASA Astrophysics Data System (ADS)

    Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

    Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

  16. The relationship between the target effective site concentration of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients

    PubMed Central

    Fan, Xiaochong; Ma, Minyu; Li, Zhisong; Gong, Shengkai; Zhang, Wei; Wen, Yuanyuan

    2015-01-01

    Objective: To study the relationship between the target effective site concentration (Ce) of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients. Methods: 50 elderly patients (ASA grade II) scheduled for selective surgical procedure under general anaesthesia were randomly divided into two groups, A and B, with 25 cases in each group. The Ce of rocuronium for intubation was 3 μg·ml-1 in both groups, and the Ce during operation were 0.8 and 1.0 μg·ml-1 in group A and B, respectively. When target controlled infusion of rocuronium was stopped, without the administration of reversal agents for neuromuscular blockade, the relationship between Ce and the first twitch height (T1) was studied by regression analysis. Results: There was a significant linear relationship between Ce and T1, and there was no statistical difference in regression coefficient and interception between group A and B (P>0.05). Conclusion: The degree of recovery from neuromuscular blockade could be judged by the target effective site concentration of rocuronium at the time of reversal from neuromuscular blockade in the elderly patients. PMID:26629159

  17. Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

    PubMed Central

    De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.

    2014-01-01

    ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411

  18. Target-site mutations conferring resistance to glyphosate in feathertop Rhodes grass (Chloris virgata) populations in Australia.

    PubMed

    Ngo, The D; Krishnan, Mahima; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

    2018-05-01

    Chloris virgata is a warm-season, C 4 , annual grass weed affecting field crops in northern Australia that has become an emerging weed in southern Australia. Four populations with suspected resistance to glyphosate were collected in South Australia, Queensland and New South Wales, Australia, and compared with one susceptible (S) population to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the rate of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in four populations of C. virgata (V12, V14.2, V14.16 and V15). GR plants were 2-9.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. GR and S plants did not differ in glyphosate absorption and translocation. Target-site EPSPS mutations corresponding to Pro-106-Leu (V14.2) and Pro-106-Ser (V15, V14.16 and V12) substitutions were found in GR populations. The population with Pro-106-Leu substitution was 2.9-4.9-fold more resistant than the three other populations with Pro-106-Ser substitution. This report confirms glyphosate resistance in C. virgata and shows that target-site EPSPS mutations confer resistance to glyphosate in this species. The evolution of glyphosate resistance in C. virgata highlights the need to identify alternative control tactics. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  19. Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

    PubMed

    Joseph, Thomas T; Osman, Roman

    2012-01-01

    In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of

  20. Kinetic analysis of the effects of target structure on siRNA efficiency

    NASA Astrophysics Data System (ADS)

    Chen, Jiawen; Zhang, Wenbing

    2012-12-01

    RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

  1. Targeting hunter distribution based on host resource selection and kill sites to manage disease risk.

    PubMed

    Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K

    2013-10-01

    Endemic and emerging diseases are rarely uniform in their spatial distribution or prevalence among cohorts of wildlife. Spatial models that quantify risk-driven differences in resource selection and hunter mortality of animals at fine spatial scales can assist disease management by identifying high-risk areas and individuals. We used resource selection functions (RSFs) and selection ratios (SRs) to quantify sex- and age-specific resource selection patterns of collared (n = 67) and hunter-killed (n = 796) nonmigratory elk (Cervus canadensis manitobensis) during the hunting season between 2002 and 2012, in southwestern Manitoba, Canada. Distance to protected area was the most important covariate influencing resource selection and hunter-kill sites of elk (AICw = 1.00). Collared adult males (which are most likely to be infected with bovine tuberculosis (Mycobacterium bovis) and chronic wasting disease) rarely selected for sites outside of parks during the hunting season in contrast to adult females and juvenile males. The RSFs showed selection by adult females and juvenile males to be negatively associated with landscape-level forest cover, high road density, and water cover, whereas hunter-kill sites of these cohorts were positively associated with landscape-level forest cover and increasing distance to streams and negatively associated with high road density. Local-level forest was positively associated with collared animal locations and hunter-kill sites; however, selection was stronger for collared juvenile males and hunter-killed adult females. In instances where disease infects a metapopulation and eradication is infeasible, a principle goal of management is to limit the spread of disease among infected animals. We map high-risk areas that are regularly used by potentially infectious hosts but currently underrepresented in the distribution of kill sites. We present a novel application of widely available data to target hunter distribution based on host resource

  2. Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

    PubMed Central

    Feinstein, P. G.; Kornfeld, K.; Hogness, D. S.; Mann, R. S.

    1995-01-01

    In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes. PMID:7498738

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

    PubMed

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

    2015-10-26

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

  4. Confirming therapeutic target of protopine using immobilized β2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

    PubMed

    Liu, Guangxin; Wang, Pei; Li, Chan; Wang, Jing; Sun, Zhenyu; Zhao, Xinfeng; Zheng, Xiaohui

    2017-07-01

    Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β 2 -adrenoceptor (β 2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β 2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β 2 -AR by the 2 methods were (1.00 ± 0.06) × 10 5 M -1 and (1.52 ± 0.14) × 10 4 M -1 . The numbers of binding sites were (1.23 ± 0.07) × 10 -7 M and (9.09 ± 0.06) × 10 -7 M, respectively. These results indicated that β 2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser 169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis. Copyright © 2017 John Wiley & Sons, Ltd.

  5. 77 FR 41807 - Dow Jones & Company, Inc., Dow Jones Content Services Division, Including On-Site Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-16

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,045; TA-W-81,045A] Dow Jones & Company, Inc., Dow Jones Content Services Division, Including On-Site Workers From Aerotek, Inc., Princeton, NJ; Generate, Inc., a Subsidiary of Dow Jones & Company, Inc., Boston, MA; Amended Certification Regarding Eligibility To Apply for Worker...

  6. 10 CFR 1021.330 - Programmatic (including site-wide) NEPA documents.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... prepare a programmatic EIS or EA (40 CFR 1502.4). DOE may also prepare a programmatic EIS or EA at any... Analysis, DOE shall determine whether the existing EIS remains adequate or whether to prepare a new site-wide EIS or supplement the existing EIS, as appropriate. The determination and supporting analysis...

  7. Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600.

    PubMed

    Wirebrand, Lisa; Madhushani, Anjana W K; Irie, Yasuhiko; Shingler, Victoria

    2018-01-01

    The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here, we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Prediction of TF target sites based on atomistic models of protein-DNA complexes

    PubMed Central

    Angarica, Vladimir Espinosa; Pérez, Abel González; Vasconcelos, Ana T; Collado-Vides, Julio; Contreras-Moreira, Bruno

    2008-01-01

    Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition. PMID:18922190

  9. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    PubMed

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. 77 FR 70478 - RG Steel Wheeling, LLC, Wheeling Office, A Division Of RG Steel, LLC, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-26

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,880: TA-A-81,880A] RG Steel Wheeling, LLC, Wheeling Office, A Division Of RG Steel, LLC, Including On-Site Leased Workers From Pro Unlimited and Green Energy Initiatives LLC, Including Workers Whose Wages Were Reported Through Severstal Wheeling, Wheeling, WV: Mountain State...

  11. Assessment of nevirapine bioavailability from targeted sites in the human gastrointestinal tract.

    PubMed

    Macha, Sreeraj; Yong, Chan-Loi; MacGregor, Thomas R; Castles, Mark; Quinson, Anne-Marie; Rouyrre, Nicolas; Wilding, Ian

    2009-12-01

    This study investigated absorption of nevirapine (NVP) from targeted sites of the gastrointestinal tract using remotely activated capsules and gamma scintigraphy. A total of 24 participants were randomized to receive 50 mg NVP orally as a suspension or via remotely activated capsules for release into the ascending colon. The 24 participants were then rerandomized into parallel groups of n = 8 for drug release into the ileum, jejunum, or descending colon. The mean gastric emptying time of capsules ranged from 0.88 to 3.35 hours. The small intestinal and colon transit time ranged from 4.08 to 7.76 hours and 17.6 to 21.2 hours, respectively, and capsule recovery time ranged from 27.6 to 34.4 hours. The relative bioavailability ratio of NVP in the jejunum was 1.06 (90% confidence interval [CI]: 1.00-1.12) compared to suspension. In the ileum, ascending colon, and descending colon, bioavailability decreased to 0.89 (0.80-0.99), 0.82 (0.71-0.95), and 0.58 (0.22-1.53), respectively. The absorption rate decreased by approximately 10-fold from the jejunum (3.83 h(-1)) to the descending colon (0.338 h(-1)), and t(max) increased from 2.42 hours (jejunum) to 16.3 hours (descending colon). Overall, NVP is absorbed from all 4 sites of the gastrointestinal tract, and the rate of absorption decreased from the jejunum to the descending colon. Relative bioavailability of NVP was in the order of jejunum > ileum > ascending colon > descending colon.

  12. Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug-target sites.

    PubMed

    Singh, Shailza; Mandlik, Vineetha; Shinde, Sonali

    2015-03-01

    GPI12 represents an important enzyme in the GPI biosynthetic pathway of several parasites like 'Leishmania'. GPI activity is generally regulated through either the hindrance in GPI complex assembly formation or the modulation of the lipophosphoglycan (LPG) flux to either reduce or enhance the pathogenicity in an organism. Of the various GPI molecules known, GPI12 is an important enzyme in the GPI biosynthetic pathway which can be exploited as a target due to the substrate specificity difference in parasites and humans. In the present study, the functional importance of the co-evolving residues of the GPI12 protein of Leishmania has been highlighted using the GPI proteins belonging to the GlcNAC-deacetylase family. Exploring the active site of the GPI12 protein and designing inhibitors against the functional residues provide ways and means to change the efficiency of deacetylation activity of the enzyme. The activity of de-N-acetylase is low in the absence of metal ions like zinc. Hence we designed eight small molecules in order to modulate the activity of GPI12. Compound 8 was found to be an appropriate choice to target the agonist (GPI12) active site thereby targeting the residues which were essential in the Zn binding and chelation activity. Inhibition of these sites offered a strong constraint to block the protein activity and in turn GPI biosynthesis.

  13. MT119, a new planar-structured compound, targets the colchicine site of tubulin arresting mitosis and inhibiting tumor cell proliferation.

    PubMed

    Zhang, Zhixiang; Meng, Tao; Yang, Na; Wang, Wei; Xiong, Bing; Chen, Yi; Ma, Lanping; Shen, Jingkang; Miao, Ze-Hong; Ding, Jian

    2011-07-01

    Microtubule-targeted drugs are now indispensable for the therapy of various cancer types worldwide. In this article, we report MT119 [6-[2-(4-methoxyphenyl) -ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido[2',1':2,3]imida-zo[4,5-c]isoquinolin-5(6H)-one] as a new microtubule-targeted agent. MT119 inhibited tubulin polymerization significantly both in tumor cells and in cell-free systems, which was followed by the disruption of mitotic spindle assembly. Surface plasmon resonance-based analyses showed that MT119 bound to purified tubulin directly, with the K(D) value of 10.6 μM. The binding of MT119 in turn caused tubulin conformational changes as evidenced by the quenched tryptophan fluorescence, the reduction of the bis-ANS reactivity and the decreased DTNB-sulfhydryl reaction rate. Competitive binding assays further revealed that MT119 bound to tubulin at its colchicine site. Consequently, by inhibiting tubulin polymerization, MT119 arrested different tumor cells at mitotic phase, which contributed to its potent antitumor activity in vitro. MT119 was also similarly cytotoxic to vincristine-, adriamycin- or mitoxantrone-resistant cancer cells and to their corresponding parental cells. Together, these data indicate that MT119 represents a new class of colchicine-site-targeted inhibitors against tubulin polymerization, which might be a promising starting point for future cancer therapeutics. Copyright © 2010 UICC.

  14. 75 FR 20386 - Auburn Hosiery Mills, Inc., Currently Known as Delta Galil, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-19

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,321] Auburn Hosiery Mills, Inc... Auburn Hosiery Mills, Inc., including on-site leased workers from Quality Personnel, Auburn, Kentucky... related to apparel. Information shows that Auburn Hosiery Mills was merged into its parent company, Delta...

  15. 75 FR 69470 - JL French Automotive Castings, LLC, Including On-Site Leased Workers From Labor Ready and Seek...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-70,143] JL French Automotive... workers of JL French Automotive Castings LLC, including on-site leased workers Labor Ready, Sheboygan... Sheboygan, Wisconsin location of JL French Automotive Castings LLC. The Department has determined that these...

  16. 77 FR 63873 - Johnson Controls, Inc. Including On-Site Leased Workers of Valley Staffing and AZ Quality Hudson...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-17

    ... workers of Johnson Controls, Inc., including on-site leased workers from Valley Staffing, Hudson..., Wisconsin location of Johnson Controls, Inc. The Department has determined that these workers were sufficiently under the control of the subject firm to be considered leased workers. Based on these findings...

  17. 78 FR 15048 - SGL Carbon, LLC Including Leased On-Site Worker of Reflex Staffing Services and Manpower, St...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-08

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-82,113] SGL Carbon, LLC Including Leased On-Site Worker of Reflex Staffing Services and Manpower, St. Marys, Pennsylvania; Notice of... of graphite parts. The initial investigation resulted in a negative determination based on the...

  18. Targeting smooth emergence: the effect site concentration of remifentanil for preventing cough during emergence during propofol-remifentanil anaesthesia for thyroid surgery.

    PubMed

    Lee, B; Lee, J-R; Na, S

    2009-06-01

    The administration of short-acting opioids can be a reliable and safe method to prevent coughing during emergence from anaesthesia but the proper dose or effect site concentration of remifentanil for this purpose has not been reported. We therefore investigated the effect site concentration (Ce) of remifentanil for preventing cough during emergence from anaesthesia with propofol-remifentanil target-controlled infusion. Twenty-three ASA I-II grade female patients, aged 23-66 yr undergoing elective thyroidectomy were enrolled in this study. EC(50) and EC(95) of remifentanil for preventing cough were determined using Dixon's up-and-down method and probit analysis. Propofol effect site concentration at extubation, mean arterial pressure, and heart rate (HR) were compared in patients with smooth emergence and without smooth emergence. Three out of 11 patients with remifentanil Ce of 1.5 ng ml(-1) and all seven patients with Ce of 2.0 ng ml(-1) did not cough during emergence; the EC(50) of remifentanil that suppressed coughing was 1.46 ng ml(-1) by Dixon's up-and-down method, and EC(95) was 2.14 ng ml(-1) by probit analysis. Effect site concentration of propofol at awakening was similar in patients with a smooth emergence and those without smooth emergence, but HR and arterial pressure were higher in those who coughed during emergence. Clinically significant hypoventilation was not seen in any patient. We found that the EC(95) of effect site concentration of remifentanil to suppress coughing at emergence from anaesthesia was 2.14 ng ml(-1). Maintaining an established Ce of remifentanil is a reliable method of abolishing cough and thereby targeting smooth emergence from anaesthesia.

  19. New Methods for Targeted Alpha Radiotherapy

    NASA Astrophysics Data System (ADS)

    Robertson, J. David

    2014-03-01

    Targeted radiotherapies based on alpha emitters are a promising alternative to beta emitting radionuclides. Because of their much shorter range, targeted α-radiotherapy (TAT) agents have great potential for application to small, disseminated tumors and micro metastases and treatment of hematological malignancies consisting of individual, circulating neoplastic cells. A promising approach to TAT is the use of the in vivo α-generator radionuclides 223 = 11.4 d) and 225Ac 1/2 = 10.0 d). In addition to their longer half-lives, these two isotopes have the potential of dramatically increasing the therapeutic efficacy of TAT as they each emit four α particles in their decay chain. This principle has recently been exploited in the development of Xofigo®, the first TAT agent approved for clinical use by the U.S. FDA. Xofigo, formulated as 223RaCl2, is used for treatment of metastatic bone cancer in men with castration-resistant prostate cancer. TAT with 223Ra works, however, only in the case of bone cancer because radium, as a chemical analogue of calcium, efficiently targets bone. In order to bring the benefits of TAT with 223Ra or 225Ac to other tumor types, a new delivery method must be devised. Retaining the in vivo α generator radionuclides at the target site through the decay process is one of the major challenges associated with the development of TAT. Because the recoil energy of the daughter radionuclides from the α-emission is ~ 100 keV - a value which is four orders of magnitude greater than the energy of a covalent bond - the daughters will not remain bound to the bioconjugate at the targeting site. Various approaches have been attempted to achieve retention of the α-generator daughter radionuclides at the target site, including incorporation of the in vivo generator into liposomes and fullerenes. Unfortunately, to date single wall liposomes and fullerenes are able to retain less than 10% of the daughter radionuclides. We have recently demonstrated that a

  20. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-07

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

  1. Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

    PubMed Central

    Korrapati, Anil Babu; Swaminathan, Gokul; Singh, Aarti; Khanna, Navin; Swaminathan, Sathyamangalam

    2012-01-01

    Background Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. Methodology/Principal Findings We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. Conclusion/Significance The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection. PMID:22848770

  2. Difference in target definition using three different methods to include respiratory motion in radiotherapy of lung cancer.

    PubMed

    Sloth Møller, Ditte; Knap, Marianne Marquard; Nyeng, Tine Bisballe; Khalil, Azza Ahmed; Holt, Marianne Ingerslev; Kandi, Maria; Hoffmann, Lone

    2017-11-01

    Minimizing the planning target volume (PTV) while ensuring sufficient target coverage during the entire respiratory cycle is essential for free-breathing radiotherapy of lung cancer. Different methods are used to incorporate the respiratory motion into the PTV. Fifteen patients were analyzed. Respiration can be included in the target delineation process creating a respiratory GTV, denoted iGTV. Alternatively, the respiratory amplitude (A) can be measured based on the 4D-CT and A can be incorporated in the margin expansion. The GTV expanded by A yielded GTV + resp, which was compared to iGTV in terms of overlap. Three methods for PTV generation were compared. PTV del (delineated iGTV expanded to CTV plus PTV margin), PTV σ (GTV expanded to CTV and A was included as a random uncertainty in the CTV to PTV margin) and PTV ∑ (GTV expanded to CTV, succeeded by CTV linear expansion by A to CTV + resp, which was finally expanded to PTV ∑ ). Deformation of tumor and lymph nodes during respiration resulted in volume changes between the respiratory phases. The overlap between iGTV and GTV + resp showed that on average 7% of iGTV was outside the GTV + resp implying that GTV + resp did not capture the tumor during the full deformable respiration cycle. A comparison of the PTV volumes showed that PTV σ was smallest and PTV Σ largest for all patients. PTV σ was in mean 14% (31 cm 3 ) smaller than PTV del , while PTV del was 7% (20 cm 3 ) smaller than PTV Σ . PTV σ yields the smallest volumes but does not ensure coverage of tumor during the full respiratory motion due to tumor deformation. Incorporating the respiratory motion in the delineation (PTV del ) takes into account the entire respiratory cycle including deformation, but at the cost, however, of larger treatment volumes. PTV Σ should not be used, since it incorporates the disadvantages of both PTV del and PTV σ .

  3. 75 FR 28655 - Rexam Closure Systems, Inc. a Subsidiary of Rexam PLC Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-21

    ..., Inc. a Subsidiary of Rexam PLC Including On-Site Leased Workers From Addeco Employment Services..., applicable to the workers of Rexam Closure Systems, Inc., a subsidiary of Rexam PLC, Hamlet, North Carolina... Rexam Closure Systems, Inc., a subsidiary of Rexam PLC purchased Owens Illinois Manufacturing. Some...

  4. 75 FR 76041 - Chrysler Group LLC Formerly Known as Chrysler LLC Kenosha Engine Plant Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... Formerly Known as Chrysler LLC Kenosha Engine Plant Including On-Site Leased Workers From Caravan Knight..., K+S Services, Inc., G4S Secure Solutions, Crassociates, Inc., CES, Inc., Evans Distribution Systems..., LLC, formerly known as Chrysler, LLC, Kenosha Engine Plant, Kenosha, Wisconsin (subject firm). The...

  5. 75 FR 20390 - Senco Brands, Inc., fka Senco Products, Inc., Including the On-Site Leased Workers of Manpower...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-19

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-70,115] Senco Brands, Inc., fka... workers of Senco Brands, Inc., fka Senco Products, Inc., including the on-site leased workers of Manpower... Brands, Inc., fka Senco Products, Inc. The Department has determined that these workers were sufficiently...

  6. 75 FR 11939 - Arkansas Lamp Manufacturing, Including On-Site Leased Workers From TEC, Van Buren, AR; Notice of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,714] Arkansas Lamp Manufacturing, Including On-Site Leased Workers From TEC, Van Buren, AR; Notice of Termination of Investigation... a petition filed on July 17, 2009, by a company official on behalf of workers of Arkansas Lamp...

  7. 75 FR 49525 - World Color (USA), LLC Formerly Known as Quebecor World World Color Covington Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-13

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,781] World Color (USA), LLC Formerly Known as Quebecor World World Color Covington Including On-Site Leased Workers From Randstad... Adjustment Assistance on May 14, 2010, applicable to workers of World Color (USA), LLC, formerly known as...

  8. Radioligand Recognition of Insecticide Targets.

    PubMed

    Casida, John E

    2018-04-04

    Insecticide radioligands allow the direct recognition and analysis of the targets and mechanisms of toxic action critical to effective and safe pest control. These radioligands are either the insecticides themselves or analogs that bind at the same or coupled sites. Preferred radioligands and their targets, often in both insects and mammals, are trioxabicyclooctanes for the γ-aminobutyric acid (GABA) receptor, avermectin for the glutamate receptor, imidacloprid for the nicotinic receptor, ryanodine and chlorantraniliprole for the ryanodine receptor, and rotenone or pyridaben for NADH + ubiquinone oxidoreductase. Pyrethroids and other Na + channel modulator insecticides are generally poor radioligands due to lipophilicity and high nonspecific binding. For target site validation, the structure-activity relationships competing with the radioligand in the binding assays should be the same as that for insecticidal activity or toxicity except for rapidly detoxified or proinsecticide analogs. Once the radioligand assay is validated for relevance, it will often help define target site modifications on selection of resistant pest strains, selectivity between insects and mammals, and interaction with antidotes and other chemicals at modulator sites. Binding assays also serve for receptor isolation and photoaffinity labeling to characterize the interactions involved.

  9. Non-target screening to trace ozonation transformation products in a wastewater treatment train including different post-treatments.

    PubMed

    Schollée, Jennifer E; Bourgin, Marc; von Gunten, Urs; McArdell, Christa S; Hollender, Juliane

    2018-05-25

    Ozonation and subsequent post-treatments are increasingly implemented in wastewater treatment plants (WWTPs) for enhanced micropollutant abatement. While this technology is effective, micropollutant oxidation leads to the formation of ozonation transformation products (OTPs). Target and suspect screening provide information about known parent compounds and known OTPs, but for a more comprehensive picture, non-target screening is needed. Here, sampling was conducted at a full-scale WWTP to investigate OTP formation at four ozone doses (2, 3, 4, and 5 mg/L, ranging from 0.3 to 1.0 gO 3 /gDOC) and subsequent changes during five post-treatment steps (i.e., sand filter, fixed bed bioreactor, moving bed bioreactor, and two granular activated carbon (GAC) filters, relatively fresh and pre-loaded). Samples were measured with online solid-phase extraction coupled to liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) using electrospray ionization (ESI) in positive and negative modes. Existing non-target screening workflows were adapted to (1) examine the formation of potential OTPs at four ozone doses and (2) compare the removal of OTPs among five post-treatments. In (1), data processing included principal component analysis (PCA) and chemical knowledge on possible oxidation reactions to prioritize non-target features likely to be OTPs. Between 394 and 1328 unique potential OTPs were detected in positive ESI for the four ozone doses tested; between 12 and 324 unique potential OTPs were detected in negative ESI. At a specific ozone dose of 0.5 gO 3 /gDOC, 27 parent compounds were identified and were related to 69 non-target features selected as potential OTPs. Two OTPs were confirmed with reference standards (venlafaxine N-oxide and chlorothiazide); 34 other potential OTPs were in agreement with literature data and/or reaction mechanisms. In (2), hierarchical cluster analysis (HCA) was applied on profiles detected in positive ESI mode across the

  10. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

    PubMed Central

    Alquezar-Planas, David E.; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N.; Lenz, Dorina; Helgen, Kristofer M.; Roca, Alfred L.; Hartman, Stefanie

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  11. Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

    PubMed

    Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria S; Allende, Miguel L; Calcaterra, Nora B

    2013-01-01

    CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.

  12. Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

    PubMed Central

    Mouguelar, Valeria S.; Allende, Miguel L.; Calcaterra, Nora B.

    2013-01-01

    CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates. PMID:23667590

  13. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP

    PubMed Central

    Hafner, Markus; Landthaler, Markus; Burger, Lukas; Khorshid, Mohsen; Hausser, Jean; Berninger, Philipp; Rothballer, Andrea; Ascano, Manuel; Jungkamp, Anna-Carina; Munschauer, Mathias; Ulrich, Alexander; Wardle, Greg S.; Dewell, Scott; Zavolan, Mihaela; Tuschl, Thomas

    2010-01-01

    Summary RNA transcripts are subject to post-transcriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases. PMID:20371350

  14. Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

    DOE PAGES

    Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; ...

    2013-12-26

    Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

  15. Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

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

    Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael

    Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

  16. Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

    PubMed Central

    Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; Akers-Rodriguez, Suzanne; Cunningham, Mark L.; Kwan, Bryan P.; Nelson, Kirk J.; Castellano, Amanda; Locke, Jeff B.; Brown-Driver, Vickie; Murphy, Timothy M.; Ong, Voon S.; Pillar, Chris M.; Shinabarger, Dean L.; Nix, Jay; Lightstone, Felice C.; Wong, Sergio E.; Nguyen, Toan B.; Shaw, Karen J.; Finn, John

    2013-01-01

    Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models. PMID:24386374

  17. 77 FR 9969 - Johnson Controls D/B/A Hoover Universal, Inc. Including On-Site Leased Workers from Kelly...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... Johnson Controls, including on-site leased workers from Kelly Services, Sycamore, Illinois. The notice was... amended notice applicable to TA-W-73,074 is hereby issued as follows: ''All workers of Johnston Controls... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,074] Johnson Controls D/B/A...

  18. 78 FR 21151 - Boise White Paper, LLC, A Subsidiary of Boise Paper Holdings, LLC, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-09

    ... Paper, LLC, A Subsidiary of Boise Paper Holdings, LLC, Including On-Site Leased Workers From Guardsmark.... Helens, OR; Boise White Paper, LLC, A Subsidiary of Boise Paper Holdings, LLC, Vancouver, WA; Amended... workers and former workers of Boise White Paper, LLC, a subsidiary of Boise Paper Holdings, LLC, St...

  19. 78 FR 40507 - Eastman Kodak Company, IPS, Including On-Site Leased Workers From Adecco, Dayton, Ohio; Notice of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-05

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-74,813A] Eastman Kodak Company... Eastman Kodak Company, IPS, including on-site leased workers from Adecco, Dayton, Ohio (TA-W-74, 813A). At the request of Eastman Kodak Company, the Department reviewed the certification applicable to workers...

  20. Algal Data from Selected Sites in the Upper Colorado River Basin, Colorado, Water Years 1996-97

    USGS Publications Warehouse

    Mize, Scott V.; Deacon, Jeffrey R.

    2001-01-01

    Algal community samples were collected at 15 sites in the Upper Colorado River Basin in Colorado as part of the National Water-Quality Assessment Program during water years 1996-97. Sites sampled were located in two physiographic provinces, the Southern Rocky Mountains and the Colorado Plateaus, and represented agricultural, mining, urban, and mixed land uses and background conditions. Algal samples were collected once per year during low-flow conditions. Quantitative algal samples were collected within two targeted instream habitat types including a taxonomically richest-targeted habitat and a depositional-targeted habitat. This report presents the algal community data collected at the fixed sites in the Upper Colorado River Basin study unit. Algal data include densities (abundance of cells per square centimeter of substrate) and biovolumes (cubic micrometers of cells per square centimeter of substrate) for the two habitat types. Quality-assurance and quality-control results for algal samples indicate that the largest sampling variability tends to occur in samples from small streams.

  1. Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network

    PubMed Central

    Joseph, Thomas T.; Osman, Roman

    2012-01-01

    In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand “seed region” have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative

  2. Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

    PubMed Central

    Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida

    2017-01-01

    Abstract Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. PMID:28053125

  3. Final Work Plan: Targeted Investigation at York, Nebraska

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

    LaFreniere, Lorraine M.

    The targeted investigation at York will be implemented in phases, so that data collected and interpretations developed at each stage of the program can be evaluated to guide subsequent phases most effectively. Section 2 of this Work Plan presents a brief overview of the York site, its geologic and hydrologic setting, and the previous CCC/USDA investigations. Section 3, outlines the proposed technical program for the targeted investigation, and Section 4 describes the investigative methods to be employed. A community relations plan is in Section 5, and Section 6 includes health and safety information. In addition to this site-specific Work Plan,more » the Master Work Plan (Argonne 2002) developed by Argonne for CCC/USDA investigations in Nebraska should be consulted for complete details of the methods and procedures to be used at York.« less

  4. 75 FR 34172 - Rexam Closure Systems, Inc., a Subsidiary of Rexam PLC, Including On-Site Leased Workers From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ..., Inc., a Subsidiary of Rexam PLC, Including On-Site Leased Workers From Addeco Employment Services and... PLC, Hamlet, North Carolina. The notice was published in the Federal Register on April 23, 2010 (75 FR... at the Hamlet, North Carolina location of Rexam Closure Systems, Inc., a subsidiary of Rexam PLC. The...

  5. 78 FR 28642 - SGL Carbon, LLC, Including Leased On-Site Worker of Reflex Staffing Services and Manpower, St...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-15

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-82,113] SGL Carbon, LLC, Including Leased On-Site Worker of Reflex Staffing Services and Manpower, St. Marys, Pennsylvania; Notice of Negative Determination on Reconsideration The initial investigation began on October 31, 2012 when a representative of the International Union of...

  6. 76 FR 50268 - Croscill Acquisition, LLC, Currently Known as Croscill Home, LLC, Plant No. 8, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-12

    ..., Currently Known as Croscill Home, LLC, Plant No. 8, Including On-Site Leased Workers From Ex-Cell Home... Fashions, a subsidiary of Croscill, Inc., Plant No. 8, Oxford, North Carolina. The notice was published in... January 14, 2011 (76 FR 2713). The workers are engaged in warehousing and distribution services of...

  7. 78 FR 39772 - Huntingdon County Site, FCI USA, LLC, Americas Division, a Subsidiary of FCI SA, Including On...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-02

    ... USA, LLC. The Department has determined that these workers were sufficiently under the control of the..., FCI USA, LLC, Americas Division, a Subsidiary of FCI SA, Including On-Site Leased Workers From... Eligibility To Apply for Worker Adjustment Assistance In accordance with Section 223 of the Trade Act of 1974...

  8. 75 FR 28657 - Dell Products LP-Parmer North Location, a Subsidiary of Dell, Inc., Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-21

    ... Spherion Corporation; Round Rock, TX; Dell Products LP--Parmer North One; Austin, TX; Amended Certification... Products LP--Parmer North Location, a Subsidiary of Dell, Inc., Including On-Site Leased Workers From... Act of 1974, as amended (``Act''), 19 U.S.C. 2273, the Department of Labor issued a Certification of...

  9. 77 FR 43122 - Corob North America, a Subsidiary of CPS Color Equipment, Inc., Including On-Site Leased Workes...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-23

    ... Subsidiary of CPS Color Equipment, Inc., Including On-Site Leased Workes From Pionear and Integra Staffing..., 2012, applicable to workers of CPS Color Equipment, Inc., formerly known as Corob North America... under the name CPS Color Equipment, Inc. The amended notice applicable to TA-W-81,222 is hereby issued...

  10. RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

    PubMed

    Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

    2006-11-21

    Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

  11. Retroviral integration: Site matters

    PubMed Central

    Demeulemeester, Jonas; De Rijck, Jan

    2015-01-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. PMID:26293289

  12. Effect of co-administration of probiotics with polysaccharide based colon targeted delivery systems to optimize site specific drug release.

    PubMed

    Prudhviraj, G; Vaidya, Yogyata; Singh, Sachin Kumar; Yadav, Ankit Kumar; Kaur, Puneet; Gulati, Monica; Gowthamarajan, K

    2015-11-01

    Significant clinical success of colon targeted dosage forms has been limited by their inappropriate release profile at the target site. Their failure to release the drug completely in the colon may be attributed to changes in the colonic milieu because of pathological state, drug effect and psychological stress accompanying the diseased state or, a combination of these. Alteration in normal colonic pH and bacterial picture leads to incomplete release of drug from the designed delivery system. We report the effectiveness of a targeted delivery system wherein the constant replenishment of the colonic microbiota is achieved by concomitant administration of probiotics along with the polysaccharide based drug delivery system. Guar gum coated spheroids of sulfasalazine were prepared. In the dissolution studies, these spheroids showed markedly higher release in the simulated colonic fluid. In vivo experiments conducted in rats clearly demonstrated the therapeutic advantage of co-administration of probiotics with guar gum coated spheroids. Our results suggest that concomitant use of probiotics along with the polysaccharide based delivery systems can be a simple strategy to achieve satisfactory colon targeting of drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site.

    PubMed

    van Bemmel, Dana M; Brank, Adam S; Eritja, Ramon; Marquez, Victor E; Christman, Judith K

    2009-09-15

    Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.

  14. DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

    PubMed Central

    van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

    2009-01-01

    Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors. PMID:19467223

  15. 75 FR 65515 - NRC Corporation, USPS Help Desk of Customer Care Center, Including On-Site Leased Workers of Volt...

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  16. 76 FR 30392 - Pisgah Yarn and Dyeing Company Including On-Site Leased Workers From Manpower, Inc. Old Fort, NC...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-25

    ... Company Including On-Site Leased Workers From Manpower, Inc. Old Fort, NC; Notice of Affirmative Determination Regarding Application for Reconsideration By application dated May 12, 2011, workers requested administrative reconsideration of the negative determination regarding workers' eligibility to apply for Trade...

  17. 77 FR 19718 - Dow Jones & Company, Inc., Dow Jones Content Services Including On-Site Workers From Aerotek, Inc...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-02

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-81,045] Dow Jones & Company, Inc., Dow Jones Content Services Including On-Site Workers From Aerotek, Inc., Princeton, NJ; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with Section 223 of the Trade Act of 1974, as amended (``Ac...

  18. 75 FR 21663 - Maysteel, LLC Including On-Site Leased Workers From Staff One, Badger Tech, Boyd Hunter, Seek...

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    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-72,483] Maysteel, LLC Including On-Site Leased Workers From Staff One, Badger Tech, Boyd Hunter, Seek, QPS, and Service First, Menomonee Falls, WI; Amended Certification Regarding Eligibility To Apply for Worker Adjustment Assistance In accordance with Section 223 of the Trade Act...

  19. 76 FR 32226 - Russell Newman, Inc., a Subsdiary of RNA Holdings, LLC, Including On-Site Leased Workers From...

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    ...,216B, TA-W-75,216C] Russell Newman, Inc., a Subsdiary of RNA Holdings, LLC, Including On-Site Leased Workers From Hour Personnel Services, Pacesetter Ontract Staffing, and Staff Force, Inc., Denton, TX; RNA...., a Subsidiary of RNA Holdings, LLC, Great Barrington, MA; RNA Holdings, LLC, a Subsidiary of SE-RN...

  20. From Bioengineering to CRISPR/Cas9 – A Personal Retrospective of 20 Years of Research in Programmable Genome Targeting

    PubMed Central

    Jeltsch, Albert

    2018-01-01

    Genome targeting of restriction enzymes and DNA methyltransferases has many important applications including genome and epigenome editing. 15–20 years ago, my group was involved in the development of approaches for programmable genome targeting, aiming to connect enzymes with an oligodeoxynucleotide (ODN), which could form a sequence-specific triple helix at the genomic target site. Importantly, the target site of such enzyme-ODN conjugate could be varied simply by altering the ODN sequence promising great applicative values. However, this approach was facing many problems including the preparation and purification of the enzyme-ODN conjugates, their efficient delivery into cells, slow kinetics of triple helix formation and the requirement of a poly-purine target site sequence. Hence, for several years genome and epigenome editing approaches mainly were based on Zinc fingers and TAL proteins as targeting devices. More recently, CRISPR/Cas systems were discovered, which use a bound RNA for genome targeting that forms an RNA/DNA duplex with one DNA strand of the target site. These systems combine all potential advantages of the once imagined enzyme-ODN conjugates and avoid all main disadvantageous. Consequently, the application of CRISPR/Cas in genome and epigenome editing has exploded in recent years. We can draw two important conclusions from this example of research history. First, evolution still is the better bioengineer than humans and, whenever tested in parallel, natural solutions outcompete engineered ones. Second, CRISPR/Cas system were discovered in pure, curiosity driven, basic research, highlighting that it is basic, bottom-up research paving the way for fundamental innovation. PMID:29434619

  1. Genome-wide analysis of Polycomb targets in Drosophila

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

    Schwartz, Yuri B.; Kahn, Tatyana G.; Nix, David A.

    2006-04-01

    Polycomb Group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. We have determined the distribution of the PcG proteins PC, E(Z) and PSC and of histone H3K27 trimethylation in the Drosophila genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb Response Elements (PREs). In contrast, H3 me3K27 forms broad domains including the entiremore » transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors but receptors, signaling proteins, morphogens and regulators representing all major developmental pathways are also included.« less

  2. Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

    PubMed

    Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

    2014-07-15

    Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

  3. Identification of new antibacterial targets in RNA polymerase of Mycobacterium tuberculosis by detecting positive selection sites.

    PubMed

    Wang, QingBiao; Xu, Yiqin; Gu, Zhuoya; Liu, Nian; Jin, Ke; Li, Yao; Crabbe, M James C; Zhong, Yang

    2018-04-01

    Bacterial RNA polymerase (RNAP) is an effective target for antibacterial treatment. In order to search new potential targets in RNAP of Mycobacterium, we detected adaptive selections of RNAP related genes in 13 strains of Mycobacterium by phylogenetic analysis. We first collected sequences of 17 genes including rpoA, rpoB, rpoC, rpoZ, and sigma factor A-M. Then maximum likelihood trees were constructed, followed by positive selection detection. We found that sigG shows positive selection along the clade (M. tuberculosis, M. bovis), suggesting its important evolutionary role and its potential to be a new antibacterial target. Moreover, the regions near 933Cys and 935His on the rpoB subunit of M. tuberculosis showed significant positive selection, which could also be a new attractive target for anti-tuberculosis drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Statistical Algorithms Accounting for Background Density in the Detection of UXO Target Areas at DoD Munitions Sites

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

    Matzke, Brett D.; Wilson, John E.; Hathaway, J.

    2008-02-12

    Statistically defensible methods are presented for developing geophysical detector sampling plans and analyzing data for munitions response sites where unexploded ordnance (UXO) may exist. Detection methods for identifying areas of elevated anomaly density from background density are shown. Additionally, methods are described which aid in the choice of transect pattern and spacing to assure with degree of confidence that a target area (TA) of specific size, shape, and anomaly density will be identified using the detection methods. Methods for evaluating the sensitivity of designs to variation in certain parameters are also discussed. Methods presented have been incorporated into the Visualmore » Sample Plan (VSP) software (free at http://dqo.pnl.gov/vsp) and demonstrated at multiple sites in the United States. Application examples from actual transect designs and surveys from the previous two years are demonstrated.« less

  5. Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

    PubMed

    Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

    2017-08-15

    MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

  6. 78 FR 52984 - Stone Age Interiors, Inc.; d/b/a Colorado Springs Marble and Granite Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-27

    ... the production of finished stone fabrication products. The workers are not separately identifiable by product line. The subject worker group includes on-site leased workers from Express Employment... articles like or directly competitive with the finished stone fabrication products produced by Stone Age...

  7. An Evaluation of a Flight Deck Interval Management Algorithm Including Delayed Target Trajectories

    NASA Technical Reports Server (NTRS)

    Swieringa, Kurt A.; Underwood, Matthew C.; Barmore, Bryan; Leonard, Robert D.

    2014-01-01

    NASA's first Air Traffic Management (ATM) Technology Demonstration (ATD-1) was created to facilitate the transition of mature air traffic management technologies from the laboratory to operational use. The technologies selected for demonstration are the Traffic Management Advisor with Terminal Metering (TMA-TM), which provides precise timebased scheduling in the terminal airspace; Controller Managed Spacing (CMS), which provides controllers with decision support tools enabling precise schedule conformance; and Interval Management (IM), which consists of flight deck automation that enables aircraft to achieve or maintain precise in-trail spacing. During high demand operations, TMA-TM may produce a schedule and corresponding aircraft trajectories that include delay to ensure that a particular aircraft will be properly spaced from other aircraft at each schedule waypoint. These delayed trajectories are not communicated to the automation onboard the aircraft, forcing the IM aircraft to use the published speeds to estimate the target aircraft's estimated time of arrival. As a result, the aircraft performing IM operations may follow an aircraft whose TMA-TM generated trajectories have substantial speed deviations from the speeds expected by the spacing algorithm. Previous spacing algorithms were not designed to handle this magnitude of uncertainty. A simulation was conducted to examine a modified spacing algorithm with the ability to follow aircraft flying delayed trajectories. The simulation investigated the use of the new spacing algorithm with various delayed speed profiles and wind conditions, as well as several other variables designed to simulate real-life variability. The results and conclusions of this study indicate that the new spacing algorithm generally exhibits good performance; however, some types of target aircraft speed profiles can cause the spacing algorithm to command less than optimal speed control behavior.

  8. Site-targeted complement inhibition by a complement receptor 2-conjugated inhibitor (mTT30) ameliorates post-injury neuropathology in mouse brains.

    PubMed

    Rich, Megan C; Keene, Chesleigh N; Neher, Miriam D; Johnson, Krista; Yu, Zhao-Xue; Ganivet, Antoine; Holers, V Michael; Stahel, Philip F

    2016-03-23

    Intracerebral complement activation after severe traumatic brain injury (TBI) leads to a cascade of neuroinflammatory pathological sequelae that propagate host-mediated secondary brain injury and adverse outcomes. There are currently no specific pharmacological agents on the market to prevent or mitigate the development of secondary cerebral insults after TBI. A novel chimeric CR2-fH compound (mTT30) provides targeted inhibition of the alternative complement pathway at the site of tissue injury. This experimental study was designed to test the neuroprotective effects of mTT30 in a mouse model of closed head injury. The administration of 500 μg mTT30 i.v. at 1 h, 4 h and 24 h after head injury attenuated complement C3 deposition in injured brains, reduced the extent of neuronal cell death, and decreased post-injury microglial activation, compared to vehicle-injected placebo controls. These data imply that site-targeted alternative pathway complement inhibition may represent a new promising therapeutic avenue for the future management of severe TBI. Copyright © 2016. Published by Elsevier Ireland Ltd.

  9. Direction, site and the muzzle target distance of bullet in the head and neck at close range as an indication of suicide or homicide.

    PubMed

    Suwanjutha, T

    1988-05-01

    Direction, site and muzzle target distance can indicate suicide or homicide. This conclusion can be drawn from autopsies of 57 cases of suicide and 68 cases of homicide by handgun fired at close range to the head and neck together with going to the crimescene in some cases. This study was carried out in Bangkok during the period from January 1983 to January 1986. In order to determine whether it was suicide or homicide, the path of the bullet, the site, the muzzle target distance must be considered. The angle of the bullet would be either elevated (from below upward), horizontal or an angle of depression (from above downward). For suicide, the direction of the bullet should be at an angle of elevation in the majority of cases. The position of the handgun in relation to the head in suicide was most often in tight contact and near contact. For homicide, the direction of the bullet should be horizontal in most cases. The bullet was at close range in the majority of the cases. There are 8 common sites for suicide and homicide and 10 different sites in the case of homicide which are at neck, left cheek, left aural region, lip, left occipital area orbit, chin, left eyebrow, submental and nose.

  10. Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

    PubMed Central

    Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

    2015-01-01

    MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

  11. Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

    PubMed

    Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

    2015-07-15

    The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

    PubMed Central

    Eggleston, Paul; Zhao, Yuguang

    2001-01-01

    Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species. PMID:11513755

  13. Satellite aerosol retrieval using dark target algorithm by coupling BRDF effect over AERONET site

    NASA Astrophysics Data System (ADS)

    Yang, Leiku; Xue, Yong; Guang, Jie; Li, Chi

    2012-11-01

    For most satellite aerosol retrieval algorithms even for multi-angle instrument, the simple forward model (FM) based on Lambertian surface assumption is employed to simulate top of the atmosphere (TOA) spectral reflectance, which does not fully consider the surface bi-directional reflectance functions (BRDF) effect. The approximating forward model largely simplifies the radiative transfer model, reduces the size of the look-up tables, and creates faster algorithm. At the same time, it creates systematic biases in the aerosol optical depth (AOD) retrieval. AOD product from the Moderate Resolution Imaging Spectro-radiometer (MODIS) data based on the dark target algorithm is considered as one of accurate satellite aerosol products at present. Though it performs well at a global scale, uncertainties are still found on regional in a lot of studies. The Lambertian surface assumpiton employed in the retrieving algorithm may be one of the uncertain factors. In this study, we first use radiative transfer simulations over dark target to assess the uncertainty to what extent is introduced from the Lambertian surface assumption. The result shows that the uncertainties of AOD retrieval could reach up to ±0.3. Then the Lambertian FM (L_FM) and the BRDF FM (BRDF_FM) are respectively employed in AOD retrieval using dark target algorithm from MODARNSS (MODIS/Terra and MODIS/Aqua Atmosphere Aeronet Subsetting Product) data over Beijing AERONET site. The validation shows that accuracy in AOD retrieval has been improved by employing the BRDF_FM accounting for the surface BRDF effect, the regression slope of scatter plots with retrieved AOD against AEROENET AOD increases from 0.7163 (for L_FM) to 0.7776 (for BRDF_FM) and the intercept decreases from 0.0778 (for L_FM) to 0.0627 (for BRDF_FM).

  14. 76 FR 35025 - Nokia, Inc.; a Subsidiary of Nokia Group; Including On-Site Leased Workers From ATC Logistics and...

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    2011-06-15

    ... of Nokia Group; Including On-Site Leased Workers From ATC Logistics and Electronics and Adecco Fort... workers from ATC Logistics and Electronics, Fort Worth, Texas. The workers supplied planning and materials... ATC Logistics and Electronics, and Adecco, Fort Worth, Texas, who became totally or partially...

  15. 78 FR 37586 - Stone Age Interiors, Inc., D/B/A Colorado Springs Marble and Granite, Including On-Site Leased...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-21

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-82,440] Stone Age Interiors, Inc., D/B/A Colorado Springs Marble and Granite, Including On-Site Leased Workers From Express Employment... Marble and Granite, Colorado Springs, Colorado (subject firm). The negative determination was issued on...

  16. A glass spherule of questionable impact origin from the Apollo 15 landing site: Unique target mare basalt

    USGS Publications Warehouse

    Ryder, G.; Delano, J.W.; Warren, P.H.; Kallemeyn, G.W.; Dalrymple, G.B.

    1996-01-01

    the young age are perhaps all most consistent with an impact origin, but nonetheless not absolutely definitive. The 15434,28 glass is distinct from the common yellow impact glasses at the Apollo 15 landing site, in particular in its lower abundances of incompatible elements and much younger age. If we accept an impact origin, then the trace element relative abundances preclude both typical KREEP and the common Apollo 15 yellow impact glass from contributing more than a few percent of the incompatible elements to potential mixtures. The melted part of any target must have consisted almost entirely of a variety (or varieties) of mare basalt or glass distinct from any known mare basalts or glasses, including Apollo 15 yellow volcanic glass, or mixtures of them. However, the rind inclusions, similar to materials of local origin, do suggest a source near the Apollo 15 landing site. An impact melt cannot have dissolved much, if any, of such inclusions. A lack of regolith materials in the rind and in the melt component suggest an immature source terrain. Thus, even for an impact origin, there is the possibility (though not requirement) that the volcanic target is younger than most mare plains. The crater Hadley C, 25 km away, is a potential source. If the 15434,28 glass is instead directly of volcanic origin, it represents an extremely young mare magma of a type previously undiscovered on the Moon.

  17. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

    PubMed

    Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D

    2017-04-07

    Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

  18. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

    PubMed Central

    Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D.

    2017-01-01

    Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification. PMID:28387220

  19. Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

    PubMed Central

    Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

    2013-01-01

    Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

  20. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    PubMed

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

  1. Identification and characterization of Hoxa9 binding sites in hematopoietic cells

    PubMed Central

    Huang, Yongsheng; Sitwala, Kajal; Bronstein, Joel; Sanders, Daniel; Dandekar, Monisha; Collins, Cailin; Robertson, Gordon; MacDonald, James; Cezard, Timothee; Bilenky, Misha; Thiessen, Nina; Zhao, Yongjun; Zeng, Thomas; Hirst, Martin; Hero, Alfred; Jones, Steven

    2012-01-01

    The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia. PMID:22072553

  2. AAVSO Target Tool: A Web-Based Service for Tracking Variable Star Observations (Abstract)

    NASA Astrophysics Data System (ADS)

    Burger, D.; Stassun, K. G.; Barnes, C.; Kafka, S.; Beck, S.; Li, K.

    2018-06-01

    (Abstract only) The AAVSO Target Tool is a web-based interface for bringing stars in need of observation to the attention of AAVSOís network of amateur and professional astronomers. The site currently tracks over 700 targets of interest, collecting data from them on a regular basis from AAVSOís servers and sorting them based on priority. While the target tool does not require a login, users can obtain visibility times for each target by signing up and entering a telescope location. Other key features of the site include filtering by AAVSO observing section, sorting by different variable types, formatting the data for printing, and exporting the data to a CSV file. The AAVSO Target Tool builds upon seven years of experience developing web applications for astronomical data analysis, most notably on Filtergraph (Burger, D., et al. 2013, Astronomical Data Analysis Software and Systems XXII, Astronomical Society of the Pacific, San Francisco, 399), and is built using the web2py web framework based on the python programming language. The target tool is available at http://filtergraph.com/aavso.

  3. Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification.

    PubMed

    Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida; Cox, J Colin; Warming, Søren

    2017-05-05

    Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Phenothiazinium based photosensitisers--photodynamic agents with a multiplicity of cellular targets and clinical applications.

    PubMed

    Harris, F; Chatfield, L K; Phoenix, D A

    2005-08-01

    PhBPs show selectivity for tumour and microbial cells, which appears to be based on electrostatic interactions between the positive charge generally carried by these molecules and the negative charge found on the outer surface of these target cells. In some cases, a site of action for photoactivated PhBPs is the outer membrane/envelope of the target cell. Such action can involve the modification of membrane lipid and/or lipopolysaccharide, and the inactivation of essential proteins and enzymes, with these effects usually leading to cell lysis and death. However, more often, PhBPs are internalised by target cells, promoted by a variety of factors, including low pH and enzymatic reduction, and upon photoactivation, internalised, PhBPs are able to inflict damage on a number of intracellular targets. In tumour cells, PhBPs can photodamage DNA and the membranes of organelles, thereby inducing necrosis and/or apoptosis. In bacterial cells, whilst DNA is generally a primary target of PhBPs, these compounds can exhibit multiple sites of action within a given cell and show different sites of action between different bacterial species. This variable targeting makes PhBPs attractive propositions as alternatives to conventional antibiotics in that the emergence of bacterial strains with acquired resistance to these compounds appears to be highly unlikely.

  5. 76 FR 27366 - Chrysler Group, LLC, Power Train Division, Mack Avenue Engine Plants #1 And #2, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-11

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-75,023] Chrysler Group, LLC, Power Train Division, Mack Avenue Engine Plants 1 And 2, Including On-Site Leased Workers from Caravan... 6, 2011, applicable to workers of Chrysler Group, LLC, Power Train Division, Mack Avenue Engine...

  6. Methods to enable the design of bioactive small molecules targeting RNA.

    PubMed

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

  7. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  8. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

    DOE PAGES

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

    2014-12-31

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

  9. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

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

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

  10. Dust deposition and removal at the MER landing sites from observations of the Panoramic Camera (Pancam) calibration targets

    NASA Astrophysics Data System (ADS)

    Kinch, K. M.; Bell, J. F.; Madsen, M. B.

    2012-12-01

    The Panoramic Cameras (Pancams) [1] on NASA's Mars Exploration Rovers have each returned in excess of 17000 images of their external calibration targets (caltargets), a set of optically well-characterized patches of materials with differing reflectance properties. During the mission dust deposition on the caltargets changed their optical reflectance properties [2]. The thickness of dust on the caltargets can be derived with high confidence from the contrast between brighter and darker colored patches. The dustier the caltarget the less contrast. We present a new history of dust deposition and removal at the two MER landing sites. Our data reveals two quite distinct dust environments. At the Spirit landing site half the Martian year is dominated by dust deposition, the other half by dust removal that usually happens during brief sharp wind events. At the Opportunity landing site the Martian year has a four-season cycle of deposition-removal-deposition-removal with dust removal happening gradually throughout the two removal seasons. Comparison to atmospheric optical depth measurements [3] shows that dust removals happen during dusty high-wind periods and that dust deposition rates are roughly proportional to the atmospheric dust load. We compare with dust deposition studies from other Mars landers and also present some early results from observation of dust on a similar camera calibration target on the Mars Science Laboratory mission. References: 1. Bell, J.F., III, et al., Mars Exploration Rover Athena Panoramic Camera (Pancam) investigation. J. Geophys. Res., 2003. 108(E12): p. 8063. 2. Kinch, K.M., et al., Dust Deposition on the Mars Exploration Rover Panoramic Camera (Pancam) Calibration Targets. J. Geophys. Res., 2007. 112(E06S03): p. doi:10.1029/2006JE002807. 3. Lemmon, M., et al., Atmospheric Imaging Results from the Mars Exploration Rovers: Spirit and Opportunity. Science, 2004. 306: p. 1753-1756. Deposited dust optical depth on the Pancam caltargets as a

  11. Smartphone-based portable wireless optical system for the detection of target analytes.

    PubMed

    Gautam, Shreedhar; Batule, Bhagwan S; Kim, Hyo Yong; Park, Ki Soo; Park, Hyun Gyu

    2017-02-01

    Rapid and accurate on-site wireless measurement of hazardous molecules or biomarkers is one of the biggest challenges in nanobiotechnology. A novel smartphone-based Portable and Wireless Optical System (PAWS) for rapid, quantitative, and on-site analysis of target analytes is described. As a proof-of-concept, we employed gold nanoparticles (GNP) and an enzyme, horse radish peroxidase (HRP), to generate colorimetric signals in response to two model target molecules, melamine and hydrogen peroxide, respectively. The colorimetric signal produced by the presence of the target molecules is converted to an electrical signal by the inbuilt electronic circuit of the device. The converted electrical signal is then measured wirelessly via multimeter in the smartphone which processes the data and displays the results, including the concentration of analytes and its significance. This handheld device has great potential as a programmable and miniaturized platform to achieve rapid and on-site detection of various analytes in a point-of-care testing (POCT) manner. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Including the online feedback site, Patient Opinion, in the nursing curriculum: Exploratory study.

    PubMed

    Jones, Ray; Young, Kim; Munro, James; Miller, Heather; Brelsford, Stephanie; Aronsson, Jennie; Goodman, Benny; Peters, Jane

    2017-10-01

    Globally, universities aim to involve people who use health services to enrich the nursing curriculum for students, but there can be barriers to this involvement. Many also want students to contribute to local communities. Online communication can help connect students to service users to achieve these aims. The online British patient feedback site, Patient Opinion, gathers comments from service users about services and encourages service responses to the comments. To explore the feasibility and acceptability of five ways of including Patient Opinion in the undergraduate nursing curriculum. Five case studies using mixed data collection methods. British University with nursing students across two campuses, accustomed to using webinars, video presentations and social media. Students from different years participated in the five approaches of making use of Patient Opinion in the curriculum; 18 students took part in an online forum to discuss Patient Opinion in the curriculum. We trialled timetabled webinars, video-linked lectures, optional enhanced access for self-study, optional audit of service user comments for two local hospitals, and optional Twitter and Tweetchat. Students discussed the aims and approaches in an online forum. Of the five approaches trialled, webinars seemed effective in ensuring that all nursing students engaged with the topic. Video-linked lectures provided an alternative when timetabling did not allow webinars, but were less interactive. The three optional approaches (Tweetchats, audit exercise, self-directed study) provided opportunities for some students to enhance their learning but students needed guidance. Sending a summary of student reviews of patients' feedback to local hospitals illustrated how students might be agents of change in local health services. Experience from these case studies suggests that webinars followed by use of Patient Opinion preparing for placements may be a sustainable way of embedding feedback sites in the

  13. Transcription factor HIF1A: downstream targets, associated pathways, polymorphic hypoxia response element (HRE) sites, and initiative for standardization of reporting in scientific literature.

    PubMed

    Slemc, Lucija; Kunej, Tanja

    2016-11-01

    Hypoxia-inducible factor-1α (HIF-1α) has crucial role in adapting cells to hypoxia through expression regulation of many genes. Identification of HIF-1α target genes (HIF-1α-TGs) is important for understanding the adapting mechanism. The aim of the present study was to collect known HIF-1α-TGs and identify their associated pathways. Targets and associated genomics data were retrieved using PubMed, WoS ( http://apps.webofknowledge.com/ ), HGNC ( http://www.genenames.org/ ), NCBI ( http://www.ncbi.nlm.nih.gov/ ), Ensemblv.84 ( http://www.ensembl.org/index.html ), DAVID Bioinformatics Resources ( https://david.ncifcrf.gov /), and Disease Ontology database ( http://disease-ontology.org/ ). From 51 papers, we collected 98 HIF-1α TGs found to be associated with 20 pathways, including metabolism of carbohydrates and pathways in cancer. Reanalysis of genomic coordinates of published HREs (hypoxia response elements) revealed six polymorphisms within HRE sites (HRE-SNPs): ABCG2, ACE, CA9, and CP. Due to large heterogeneity of results presentation in scientific literature, we also propose a first step towards reporting standardization of HIF-1α-target interactions consisting of ten relevant data types. Suggested minimal checklist for reporting will enable faster development of a complete catalog of HIF-1α-TGs, data sharing, bioinformatics analyses, and setting novel more targeted hypotheses. The proposed format for data standardization is not yet complete but presents a baseline for further optimization of the protocol with additional details, for example, regarding the experimental validation.

  14. A tale of two sequences: microRNA-target chimeric reads.

    PubMed

    Broughton, James P; Pasquinelli, Amy E

    2016-04-04

    In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

  15. Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2013-11-01

    The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

  16. Downregulation of miR-29a/b/c in placenta accreta inhibits apoptosis of implantation site intermediate trophoblast cells by targeting MCL1.

    PubMed

    Gu, Yongzhong; Bian, Yuehong; Xu, Xiaofei; Wang, Xietong; Zuo, Changting; Meng, Jinlai; Li, Hongyan; Zhao, Shigang; Ning, Yunnan; Cao, Yongzhi; Huang, Tao; Yan, Junhao; Chen, Zi-Jiang

    2016-12-01

    Placenta accreta is defined as abnormal adhesion of placental villi to the uterine myometrium. Although this condition has become more common as a result of the increasing rate of cesarean sections, the underlying causative mechanism(s) remain elusive. Because microRNA-29a/b/c (miR-29a/b/c) have been shown to play important roles in placental development, this study evaluated the roles of these microRNAs in placenta accreta. Expression of miR-29a/b/c and myeloid cell leukemia-1 (MCL1) were quantified in patient tissues and HTR8/SVneo trophoblast cells using the real-time quantitative polymerase chain reaction. Western blotting was used to analyze expression of the MCL1 protein in HTR8/SVneo trophoblast cells with altered expression of miR-29a/b/c. To determine their role in apoptosis, miR-29a/b/c were overexpressed in HTR-8/SVneo cells, and levels of apoptosis were analyzed by flow cytometry. Luciferase activity assays were used to determine whether MCL1 is a target gene of miR-29a/b/c. Expression of miR-29a/b/c was significantly lower in creta sites compared to noncreta sites (p = 0.018, 0.041, and 0.022, respectively), but expression of MCL1 was upregulated in creta sites (p = 0.039). MCL1 expression was significantly downregulated in HTR-8/SVneo cells overexpressing miR-29a/b/c (p = 0.002, 0.008, and 0.013, respectively). Luciferase activity assays revealed that miR-29a/b/c directly target the 3' untranslated region of MCL1 in 293T cells. Over-expression of miR-29a/b/c induced apoptosis in the HTR-8/SVneo trophoblast cell line. Moreover, histopathological evaluation revealed that the number of implantation site intermediate trophoblast (ISIT) cells was increased in creta sites and that these cells were positive for MCL1. Our results demonstrate that in placenta accreta, miR-29a/b/c inhibits apoptosis of ISIT cells by targeting MCL1. These findings provide new insights into the pathogenesis of placenta accreta. Copyright © 2016 Elsevier Ltd. All rights

  17. Instrumentation of Molecular Imaging on Site-Specific Targeting Fluorescent Peptide for Early Detection of Breast Cancer

    NASA Astrophysics Data System (ADS)

    Yu, Ping; Ma, Lixin

    2012-02-01

    In this work we developed two biomedical imaging techniques for early detection of breast cancer. Both image modalities provide molecular imaging capability to probe site-specific targeting dyes. The first technique, heterodyne CCD fluorescence mediated tomography, is a non-invasive biomedical imaging that uses fluorescent photons from the targeted dye on the tumor cells inside human breast tissue. The technique detects a large volume of tissue (20 cm) with a moderate resolution (1 mm) and provides the high sensitivity. The second technique, dual-band spectral-domain optical coherence tomography, is a high-resolution tissue imaging modality. It uses a low coherence interferometer to detect coherent photons hidden in the incoherent background. Due to the coherence detection, a high resolution (20 microns) is possible. We have finished prototype imaging systems for the development of both image modalities and performed imaging experiments on tumor tissues. The spectroscopic/tomographic images show contrasts of dense tumor tissues and tumor necrotic regions. In order to correlate the findings from our results, a diffusion-weighted magnetic resonance imaging (MRI) of the tumors was performed using a small animal 7-Telsa MRI and demonstrated excellent agreement.

  18. Self-assembled peptide-based nanostructures: Smart nanomaterials toward targeted drug delivery.

    PubMed

    Habibi, Neda; Kamaly, Nazila; Memic, Adnan; Shafiee, Hadi

    2016-02-01

    Self-assembly of peptides can yield an array of well-defined nanostructures that are highly attractive nanomaterials for many biomedical applications such as drug delivery. Some of the advantages of self-assembled peptide nanostructures over other delivery platforms include their chemical diversity, biocompatibility, high loading capacity for both hydrophobic and hydrophilic drugs, and their ability to target molecular recognition sites. Furthermore, these self-assembled nanostructures could be designed with novel peptide motifs, making them stimuli-responsive and achieving triggered drug delivery at disease sites. The goal of this work is to present a comprehensive review of the most recent studies on self-assembled peptides with a focus on their "smart" activity for formation of targeted and responsive drug-delivery carriers.

  19. Hanford Site Environmental Safety and Health Fiscal Year 2001 Budget-Risk management summary

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

    REEP, I.E.

    1999-05-12

    The Hanford Site Environment, Safety and Health (ES&H) Budget-Risk Management Summary report is prepared to support the annual request to sites in the U.S. Department of Energy (DOE) Complex by DOE, Headquarters. The request requires sites to provide supplementary crosscutting information related to ES&H activities and the ES&H resources that support these activities. The report includes the following: (1) A summary status of fiscal year (FY) 1999 ES&H performance and ES&H execution commitments; (2)Status and plans of Hanford Site Office of Environmental Management (EM) cleanup activities; (3) Safety and health (S&H) risk management issues and compliance vulnerabilities of FY 2001more » Target Case and Below Target Case funding of EM cleanup activities; (4) S&H resource planning and crosscutting information for FY 1999 to 2001; and (5) Description of indirect-funded S&H activities.« less

  20. Ablation of post-surgical intra-atrial reentrant tachycardia. Predilection target sites and mapping approach.

    PubMed

    Anné, W; van Rensburg, H; Adams, J; Ector, H; Van de Werf, F; Heidbüchel, H

    2002-10-01

    Atrial arrhythmias are a frequent complication of atrial surgery. The location of these tachycardias is very diverse due to the individual difference in the original anatomy, surgical corrections, and effects of atrial fibrosis. Nevertheless some recurrent patterns are emerging. Forty-five patients underwent 51 ablation procedures between September 1995 and March 2001 using conventional mapping and temperature-controlled ablation. A duadecapolar catheter was swept from anterior to posterior in the right (and/or left) atrium, allowing for rapid mapping followed by entrainment confirmation. Twenty-eight patients had corrected congenital heart disease, 17 surgery for acquired heart disease. One hundred and sixteen arrhythmias were found, 86 circuits were targeted, 81 with success (94%). Despite the heterogeneous anatomy, the same targets were often encountered: the posterior isthmus between the inferior vena cava and the tricuspid ring (62%), the gap between the inferior vena cava and the atriotomy scar (49%), and the region around the atriopulmonary connection in Fontans (two out of four patients). After a mean follow-up of 24 months, 13 patients had a recurrent arrhythmia (29%) after their last procedure. There was a significant association between the number of circuits found during the initial procedure and the likelihood of recurrent arrhythmias. Knowledge of anatomical predilection sites and mapping the right (and/or left) atrium with a 'sweeping Halo technique' allow for effective ablation of most post-surgical atrial tachycardias. Severely damaged atria with multiple arrhythmias may require 'preventive' ablation of all recognizable channels.

  1. Vascular targeting of a gold nanoparticle to breast cancer metastasis

    PubMed Central

    Peiris, Pubudu M.; Deb, Partha; Doolittle, Elizabeth; Doron, Gilad; Goldberg, Amy; Govender, Priya; Shah, Shruti; Rao, Swetha; Carbone, Sarah; Cotey, Thomas; Sylvestre, Meilyn; Singh, Sohaj; Schiemann, William P.; Lee, Zhenghong; Karathanasis, Efstathios

    2015-01-01

    The vast majority of breast cancer deaths are due to metastatic disease. While deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the gold nanoparticles, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Due to the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles. PMID:26036431

  2. BEAM ON TARGET MODEL Produces All Gamma Ray Burst Phenomena Including Afterglow

    NASA Astrophysics Data System (ADS)

    Greyber, H.

    2000-12-01

    While one must applaud the splendid research by L. Piro et al and L. Amati et al reported in SCIENCE recently, one must question, as M. Rees and S. Woolsey have done, their conclusion that a ``supranova model" is the only explanation for these new X-ray observations. In fact L. Piro was quoted as saying, ``Our data helps rule out the scenario where two neutron stars or black holes collide. We think GRBs result from something similar to a supernova explosion, but much more powerful." A relatively unknown physical model for GRBs, Greyber's Beam On Target model (BOT), dating back to the first CGRO observations, can plausibly explain the iron emission lines observed for GRB991216, and also the mass of the dense medium within a light-day of the GRB being roughly equivalent to at least one-tenth solar mass, as well as the initial shedding of material followed by the GRB event. When a galaxy forms under gravitational collapse in the presence of a primordial magnetic field, Mestel and Strittmatter demonstrated that, for finite Ohmic diffusion, a growing equatorial current loop is formed. Even if this stable ``Storage Ring" has only 10exp-9 of the total energy released during a typical galaxy's formation, the relativistic beam can possess 10exp58 ergs. The GRB ``fireball" occurs when a target star races across the powerful beam, blowing off target material as a hot, rapidly expanding plasma cloud, simulating an explosion. Since currents in space are known to be sometimes filamentary, sharp millisecond spikes can be expected in some GRBs. Proton and alpha particle nuclear reactions produce a gamma ray beam. Beam particles impinging on denser cloud material create an electromagnetic shower, producing X-ray, optical and radio radiation. Since the Storage Ring has an intense magnetic field around it, synchrotron radiation is expected. The beam, striking a highly evolved massive target star, produces the iron emission lines. H. D. Greyber, in ``After the Dark Ages:When Galaxies

  3. Occurrence of target-site resistance to neonicotinoids in the aphid Myzus persicae in Tunisia, and its status on different host plants.

    PubMed

    Charaabi, Kamel; Boukhris-Bouhachem, Sonia; Makni, Mohamed; Denholm, Ian

    2018-06-01

    The R81T mutation conferring target-site resistance to neonicotinoid insecticides in Myzus persicae was first detected in France and has since spread across much of southern Europe. In response to recent claims of control failure with neonicotinoids in Tunisia, we have used a molecular assay to investigate the presence and distribution of this target-site mutation in samples collected from six locations and six crops attacked by M. persicae. The resistance allele containing R81T was present at substantial frequencies (32-55%) in aphids collected between 2014 and 2016 from northern Tunisia but was much rarer further south. It occurred in aphids collected from the aphid's primary host (peach) and four secondary crop hosts (potato, pepper, tomato and melon). Its absence in aphids from tobacco highlights complexities in the systematics of M. persicae that require further investigation. This first report of R81T from North Africa reflects a continuing expansion of its range around the Mediterranean Basin, although it remains unrecorded elsewhere in the world. Loss of efficacy of neonicotinoids presents a serious threat to the sustainability of aphid control. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  4. Lipid Nanoparticles: A novel approach for brain targeting.

    PubMed

    Shankar, Ravi; Joshi, Monika; Pathak, Kamla

    2018-06-10

    Brain is a delicate organ, separated from general circulation and is characterized by the presence of relatively impermeable Blood Brain Barrier (BBB). The BBB maintains homeostasis in the brain thus restricting the entrance of foreign bodies and several molecules from reaching the brain. As a result several promising molecules do not reach the target site and fail to produce in vivo response. Nevertheless, lipid nanoparticles are taken up readily by the brain because of their lipophilic nature. The bioacceptable and biodegradable nature of lipid nanoparticles makes them less toxic and suited for brain targeting. In the present review the BBB, mechanism of transport across the BBB, strategies to bypass the blood-brain barrier have been presented. The aptness of lipid nanoparticles for brain targeting has been highlighted. The proposed mechanism of uptake of the lipid nanoparticles, methods of prolonging the plasma retention and various methods of preparation for formulation of effective delivery systems for brain targeting have been included and dealt in this review. Lipid based formulations can be designated as the current and future generation of drug delivery systems as these possess tremendous potential to bypass BBB and reach the target site due to their small size and ability to dodge the reticular endothelial system. However, these nanostructures need to be investigated intensively to successfully reach the clinical trials stage. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Humoral immunity targeting site I of antigenic domain 2 of glycoprotein B upon immunization with different cytomegalovirus candidate vaccines.

    PubMed

    Axelsson, Fredrika; Adler, Stuart P; Lamarre, Alain; Ohlin, Mats

    2007-12-21

    Glycoprotein B (gB) is a major component in several vaccines that are under development for prevention of disease by cytomegalovirus. It contains multiple determinants that are targets for neutralizing antibodies. One of them is site I of antigenic domain 2 (AD-2). The epitope, defined by short peptides, is quite conserved between different isolates. However, it is poorly immunogenic in natural infection. In this study we investigated the extent to which different vaccines, attenuated live Towne vaccine with or without priming with a canarypox virus coding for gB, or a recombinant gB vaccine adjuvanted with MF59, induced antibodies to this epitope. As in natural infection only a fraction of all subjects developed antibody responses against site I of AD-2 following vaccination. We suggest that strategies that enhance immunogenicity of this epitope will improve vaccine efficacy.

  6. Low Herbivory among Targeted Reforestation Sites in the Andean Highlands of Southern Ecuador

    PubMed Central

    Adams, Marc-Oliver; Fiedler, Konrad

    2016-01-01

    Insect herbivory constitutes an important constraint in the viability and management of targeted reforestation sites. Focusing on young experimental stands at about 2000 m elevation in southern Ecuador, we examined foliar damage over one season as a function of tree species and habitat. Native tree species (Successional hardwood: Cedrela montana and Tabebuia chrysantha; fast-growing pioneer: Heliocarpus americanus) have been planted among prevailing local landcover types (abandoned pasture, secondary shrub vegetation, and a Pinus patula plantation) in 2003/4. Plantation trees were compared to conspecifics in the spontaneous undergrowth of adjacent undisturbed rainforest matched for height and foliar volume. Specifically, we tested the hypotheses that H. americanus as a pioneer species suffers more herbivory compared to the two successional tree species, and that damage is inversely related to habitat complexity. Overall leaf damage caused by folivorous insects (excluding leafcutter ants) was low. Average leaf loss was highest among T. chrysantha (7.50% ± 0.19 SE of leaf area), followed by H. americanus (4.67% ± 0.18 SE) and C. montana (3.18% ± 0.15 SE). Contrary to expectations, leaf area loss was highest among trees in closed-canopy natural rainforest, followed by pine plantation, pasture, and secondary shrub sites. Harvesting activity of leafcutter ants (Acromyrmex sp.) was strongly biased towards T. chrysantha growing in open habitat (mean pasture: 2.5%; shrub: 10.5%) where it could result in considerable damage (> 90.0%). Insect folivory is unlikely to pose a barrier for reforestation in the tropical Andean mountain forest zone at present, but leafcutter ants may become problematic if local temperatures increase in the wake of global warming. PMID:26963395

  7. Low Herbivory among Targeted Reforestation Sites in the Andean Highlands of Southern Ecuador.

    PubMed

    Adams, Marc-Oliver; Fiedler, Konrad

    2016-01-01

    Insect herbivory constitutes an important constraint in the viability and management of targeted reforestation sites. Focusing on young experimental stands at about 2000 m elevation in southern Ecuador, we examined foliar damage over one season as a function of tree species and habitat. Native tree species (Successional hardwood: Cedrela montana and Tabebuia chrysantha; fast-growing pioneer: Heliocarpus americanus) have been planted among prevailing local landcover types (abandoned pasture, secondary shrub vegetation, and a Pinus patula plantation) in 2003/4. Plantation trees were compared to conspecifics in the spontaneous undergrowth of adjacent undisturbed rainforest matched for height and foliar volume. Specifically, we tested the hypotheses that H. americanus as a pioneer species suffers more herbivory compared to the two successional tree species, and that damage is inversely related to habitat complexity. Overall leaf damage caused by folivorous insects (excluding leafcutter ants) was low. Average leaf loss was highest among T. chrysantha (7.50% ± 0.19 SE of leaf area), followed by H. americanus (4.67% ± 0.18 SE) and C. montana (3.18% ± 0.15 SE). Contrary to expectations, leaf area loss was highest among trees in closed-canopy natural rainforest, followed by pine plantation, pasture, and secondary shrub sites. Harvesting activity of leafcutter ants (Acromyrmex sp.) was strongly biased towards T. chrysantha growing in open habitat (mean pasture: 2.5%; shrub: 10.5%) where it could result in considerable damage (> 90.0%). Insect folivory is unlikely to pose a barrier for reforestation in the tropical Andean mountain forest zone at present, but leafcutter ants may become problematic if local temperatures increase in the wake of global warming.

  8. Targeted left ventricular lead placement to guide cardiac resynchronization therapy: the TARGET study: a randomized, controlled trial.

    PubMed

    Khan, Fakhar Z; Virdee, Mumohan S; Palmer, Christopher R; Pugh, Peter J; O'Halloran, Denis; Elsik, Maros; Read, Philip A; Begley, David; Fynn, Simon P; Dutka, David P

    2012-04-24

    This study sought to assess the impact of targeted left ventricular (LV) lead placement on outcomes of cardiac resynchronization therapy (CRT). Placement of the LV lead to the latest sites of contraction and away from the scar confers the best response to CRT. We conducted a randomized, controlled trial to compare a targeted approach to LV lead placement with usual care. A total of 220 patients scheduled for CRT underwent baseline echocardiographic speckle-tracking 2-dimensional radial strain imaging and were then randomized 1:1 into 2 groups. In group 1 (TARGET [Targeted Left Ventricular Lead Placement to Guide Cardiac Resynchronization Therapy]), the LV lead was positioned at the latest site of peak contraction with an amplitude of >10% to signify freedom from scar. In group 2 (control) patients underwent standard unguided CRT. Patients were classified by the relationship of the LV lead to the optimal site as concordant (at optimal site), adjacent (within 1 segment), or remote (≥2 segments away). The primary endpoint was a ≥15% reduction in LV end-systolic volume at 6 months. Secondary endpoints were clinical response (≥1 improvement in New York Heart Association functional class), all-cause mortality, and combined all-cause mortality and heart failure-related hospitalization. The groups were balanced at randomization. In the TARGET group, there was a greater proportion of responders at 6 months (70% vs. 55%, p = 0.031), giving an absolute difference in the primary endpoint of 15% (95% confidence interval: 2% to 28%). Compared with controls, TARGET patients had a higher clinical response (83% vs. 65%, p = 0.003) and lower rates of the combined endpoint (log-rank test, p = 0.031). Compared with standard CRT treatment, the use of speckle-tracking echocardiography to the target LV lead placement yields significantly improved response and clinical status and lower rates of combined death and heart failure-related hospitalization. (Targeted Left Ventricular Lead

  9. Methods to enable the design of bioactive small molecules targeting RNA

    PubMed Central

    Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

    2014-01-01

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  10. Evolutionary diversity and potential recombinogenic role of integration targets of non-LTR retrotransposons

    PubMed Central

    Gentles, Andrew J.; Kohany, Oleksiy; Jurka, Jerzy

    2005-01-01

    Short interspersed elements (SINEs) make up a significant fraction of total DNA in mammalian genomes, providing a rich substrate for chromosomal rearrangements by SINE-SINE recombinations. Proliferation of mammalian SINEs is mediated primarily by LINE1 (L1) non-LTR retrotransposons that preferentially integrate at DNA sequence targets with average length ~15 bp and containing conserved endonucleolytic nicking signals at both ends. We report that sequence variations in the first of the two nicking signals, represented by a 5′TT-AAAA consensus sequence, affect the position of the second signal thus leading to target site duplications (TSDs) of different lengths. The length distribution of TSDs appears to be affected also by L1-encoded enzyme variants, since targets with the same 5′ nicking site can be of different average length in different mammalian species. Taking this into account, we re-analyzed the second nicking site and found that it is larger and includes more conserved sites than previously appreciated, with a consensus of 5′ANTNTN-AA. We also studied potential involvement of the nicking sites in stimulating recombinations between SINE elements. We determined that SINE elements retaining TSDs with perfect 5′TT-AAAA nicking sites appear to be lost relatively rapidly from the human and rat genomes, and less rapidly from dog. We speculate that the introduction of single-strand DNA breaks induced by recurring endonucleolytic attacks at these sites, combined with the ubiquitousness of SINEs, may significantly promote recombination between repetitive elements, leading to the observed losses. At the same time new L1 subfamilies may be selected for “incompatibility” with pre-existing targets. This provides a possible driving force for the continual emergence of new L1 subfamilies which, in turn, may affect selection of L1-dependent SINE subfamilies. PMID:15944437

  11. Site characterization at the Rabbit Valley Geophysical Performance Evaluation Range

    NASA Astrophysics Data System (ADS)

    Koppenjan, S.,; Martinez, M.

    The United States Department of Energy (US DOE) is developing a Geophysical Performance Evaluation Range (GPER) at Rabbit Valley located 30 miles west of Grand Junction, Colorado. The purpose of the range is to provide a test area for geophysical instruments and survey procedures. Assessment of equipment accuracy and resolution is accomplished through the use of static and dynamic physical models. These models include targets with fixed configurations and targets that can be re-configured to simulate specific specifications. Initial testing (1991) combined with the current tests at the Rabbit Valley GPER will establish baseline data and will provide performance criteria for the development of geophysical technologies and techniques. The US DOE's Special Technologies Laboratory (STL) staff has conducted a Ground Penetrating Radar (GPR) survey of the site with its stepped FM-CW GPR. Additionally, STL contracted several other geophysical tests. These include an airborne GPR survey incorporating a 'chirped' FM-CW GPR system and a magnetic survey with a surfaced-towed magnetometer array unit Ground-based and aerial video and still frame pictures were also acquired. STL compiled and analyzed all of the geophysical maps and created a site characterization database. This paper discusses the results of the multi-sensor geophysical studies performed at Rabbit Valley and the future plans for the site.

  12. Vascular Targeting of a Gold Nanoparticle to Breast Cancer Metastasis.

    PubMed

    Peiris, Pubudu M; Deb, Partha; Doolittle, Elizabeth; Doron, Gilad; Goldberg, Amy; Govender, Priya; Shah, Shruti; Rao, Swetha; Carbone, Sarah; Cotey, Thomas; Sylvestre, Meilyn; Singh, Sohaj; Schiemann, William P; Lee, Zhenghong; Karathanasis, Efstathios

    2015-08-01

    The vast majority of breast cancer deaths are due to metastatic disease. Although deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle (AuNP) to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the AuNPs, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Because of the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  13. Containment wells to form hydraulic barriers along site boundaries.

    PubMed

    Vo, D; Ramamurthy, A S; Qu, J; Zhao, X P

    2008-12-15

    In the field, aquifer remediation methods include pump and treat procedures based on hydraulic control systems. They are used to reduce the level of residual contamination present in the soil and soil pores of aquifers. Often, physical barriers are erected along the boundaries of the target (aquifer) site to reduce the leakage of the released soil contaminant to the surrounding regions. Physical barriers are expensive to build and dismantle. Alternatively, based on simple hydraulic principles, containment wells or image wells injecting clear water can be designed and built to provide hydraulic barriers along the contaminated site boundaries. For brevity, only one pattern of containment well system that is very effective is presented in detail. The study briefly reports about the method of erecting a hydraulic barrier around a contaminated region based on the simple hydraulic principle of images. During the clean-up period, hydraulic barriers can considerably reduce the leakage of the released contaminant from the target site to surrounding pristine regions. Containment wells facilitate the formation of hydraulic barriers. Hence, they control the movement of contaminants away from the site that is being remedied. However, these wells come into play, only when the pumping operation for cleaning up the site is active. After operation, they can be filled with soil to permit the natural ground water movement. They can also be used as monitoring wells.

  14. Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications.

    PubMed

    Christensen, S; Pont-Kingdon, G; Carroll, D

    2000-01-01

    In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non-cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed - that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data.

  15. Electronic Cigarette Marketing Online: a Multi-Site, Multi-Product Comparison.

    PubMed

    Chu, Kar-Hai; Sidhu, Anupreet K; Valente, Thomas W

    2015-01-01

    Electronic cigarette awareness and use has been increasing rapidly. E-cigarette brands have utilized social networking sites to promote their products, as the growth of the e-cigarette industry has paralleled that of Web 2.0. These online platforms are cost-effective and have unique technological features and user demographics that can be attractive for selective marketing. The popularity of multiple sites also poses a risk of exposure to social networks where e-cigarette brands might not have a presence. To examine the marketing strategies of leading e-cigarette brands on multiple social networking sites, and to identify how affordances of the digital media are used to their advantage. Secondary analyses include determining if any brands are benefitting from site demographics, and exploring cross-site diffusion of marketing content through multi-site users. We collected data from two e-cigarette brands from four social networking sites over approximately 2.5 years. Content analysis is used to search for themes, population targeting, marketing strategies, and cross-site spread of messages. Twitter appeared to be the most frequently used social networking site for interacting directly with product users. Facebook supported informational broadcasts, such as announcements regarding political legislation. E-cigarette brands also differed in their approaches to their users, from informal conversations to direct product marketing. E-cigarette makers use different strategies to market their product and engage their users. There was no evidence of direct targeting of vulnerable populations, but the affordances of the different sites are exploited to best broadcast context-specific messages. We developed a viable method to study cross-site diffusion, although additional refinement is needed to account for how different types of digital media are used.

  16. CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.

    PubMed

    Miki, Daisuke; Zhang, Wenxin; Zeng, Wenjie; Feng, Zhengyan; Zhu, Jian-Kang

    2018-05-17

    Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

  17. The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis

    PubMed Central

    2012-01-01

    Background The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. Results In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. Conclusion The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences. PMID:22436504

  18. SuperTarget goes quantitative: update on drug–target interactions

    PubMed Central

    Hecker, Nikolai; Ahmed, Jessica; von Eichborn, Joachim; Dunkel, Mathias; Macha, Karel; Eckert, Andreas; Gilson, Michael K.; Bourne, Philip E.; Preissner, Robert

    2012-01-01

    There are at least two good reasons for the on-going interest in drug–target interactions: first, drug-effects can only be fully understood by considering a complex network of interactions to multiple targets (so-called off-target effects) including metabolic and signaling pathways; second, it is crucial to consider drug-target-pathway relations for the identification of novel targets for drug development. To address this on-going need, we have developed a web-based data warehouse named SuperTarget, which integrates drug-related information associated with medical indications, adverse drug effects, drug metabolism, pathways and Gene Ontology (GO) terms for target proteins. At present, the updated database contains >6000 target proteins, which are annotated with >330 000 relations to 196 000 compounds (including approved drugs); the vast majority of interactions include binding affinities and pointers to the respective literature sources. The user interface provides tools for drug screening and target similarity inclusion. A query interface enables the user to pose complex queries, for example, to find drugs that target a certain pathway, interacting drugs that are metabolized by the same cytochrome P450 or drugs that target proteins within a certain affinity range. SuperTarget is available at http://bioinformatics.charite.de/supertarget. PMID:22067455

  19. Novel Targeted Therapies for Inflammatory Bowel Disease.

    PubMed

    Coskun, Mehmet; Vermeire, Severine; Nielsen, Ole Haagen

    2017-02-01

    Our growing understanding of the immunopathogenesis of inflammatory bowel disease (IBD) has opened new avenues for developing targeted therapies. These advances in treatment options targeting different mechanisms of action offer new hope for personalized management. In this review we highlight emerging novel and easily administered therapeutics that may be viable candidates for the management of IBD, such as antibodies against interleukin 6 (IL-6) and IL-12/23, small molecules including Janus kinase inhibitors, antisense oligonucleotide against SMAD7 mRNA, and inhibitors of leukocyte trafficking to intestinal sites of inflammation (e.g., sphingosine 1-phosphate receptor modulators). We also provide an update on the current status in clinical development of these new classes of therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. RNA-guided genome editing for target gene mutations in wheat.

    PubMed

    Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

    2013-12-09

    The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.