Sample records for additional site specific

  1. Site Preference of Ternary Alloying Additions to AuTi

    NASA Technical Reports Server (NTRS)

    Bozzolo, Guillermo; Mosca, Hugo O.; Noebe, Ronald D.

    2006-01-01

    Atomistic modeling of the site substitution behavior of several alloying additions, namely. Na, Mg, Al, Si. Sc, V, Cr, Mn. Fe, Co, Ni, Cu, Zn, Y, Zr. Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, and Pt in B2 TiAu is reported. The 30 elements can be grouped according to their absolute preference for a specific site, regardless of concentration, or preference for available sites in the deficient sublattice. Results of large scale simulations are also presented, distinguishing between additions that remain in solution from those that precipitate a second phase.

  2. Quantifying site-specific physical heterogeneity within an estuarine seascape

    USGS Publications Warehouse

    Kennedy, Cristina G.; Mather, Martha E.; Smith, Joseph M.

    2017-01-01

    Quantifying physical heterogeneity is essential for meaningful ecological research and effective resource management. Spatial patterns of multiple, co-occurring physical features are rarely quantified across a seascape because of methodological challenges. Here, we identified approaches that measured total site-specific heterogeneity, an often overlooked aspect of estuarine ecosystems. Specifically, we examined 23 metrics that quantified four types of common physical features: (1) river and creek confluences, (2) bathymetric variation including underwater drop-offs, (3) land features such as islands/sandbars, and (4) major underwater channel networks. Our research at 40 sites throughout Plum Island Estuary (PIE) provided solutions to two problems. The first problem was that individual metrics that measured heterogeneity of a single physical feature showed different regional patterns. We solved this first problem by combining multiple metrics for a single feature using a within-physical feature cluster analysis. With this approach, we identified sites with four different types of confluences and three different types of underwater drop-offs. The second problem was that when multiple physical features co-occurred, new patterns of total site-specific heterogeneity were created across the seascape. This pattern of total heterogeneity has potential ecological relevance to structure-oriented predators. To address this second problem, we identified sites with similar types of total physical heterogeneity using an across-physical feature cluster analysis. Then, we calculated an additive heterogeneity index, which integrated all physical features at a site. Finally, we tested if site-specific additive heterogeneity index values differed for across-physical feature clusters. In PIE, the sites with the highest additive heterogeneity index values were clustered together and corresponded to sites where a fish predator, adult striped bass (Morone saxatilis), aggregated in a

  3. Nanoparticles for Site Specific Genome Editing

    NASA Astrophysics Data System (ADS)

    McNeer, Nicole Ali

    translate gene therapies for diseases of the blood and immune system to clinical practice. In addition, we have expanded the use of this technology to an additional nonhematopoietic model system: correction of the human cystic fibrosis transmembrane receptor gene in human bronchial epithelial cells. The work presented here represents (1) the first use of biodegradable nanoparticles for PNA delivery, (2) the first direct in vivo site-specific genome modification in human cells, and (3) the first use of triplex-PNA technology for site-specific genome editing in cystic fibrosis.

  4. 40 CFR 228.6 - Specific criteria for site selection.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Specific criteria for site selection... selection. (a) In the selection of disposal sites, in addition to other necessary or appropriate factors...) Existence at or in close proximity to the site of any significant natural or cultural features of historical...

  5. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

    PubMed Central

    McClelland, M; Nelson, M; Raschke, E

    1994-01-01

    Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074

  6. TSAPA: identification of tissue-specific alternative polyadenylation sites in plants.

    PubMed

    Ji, Guoli; Chen, Moliang; Ye, Wenbin; Zhu, Sheng; Ye, Congting; Su, Yaru; Peng, Haonan; Wu, Xiaohui

    2018-06-15

    Alternative polyadenylation (APA) is now emerging as a widespread mechanism modulated tissue-specifically, which highlights the need to define tissue-specific poly(A) sites for profiling APA dynamics across tissues. We have developed an R package called TSAPA based on the machine learning model for identifying tissue-specific poly(A) sites in plants. A feature space including more than 200 features was assembled to specifically characterize poly(A) sites in plants. The classification model in TSAPA can be customized by selecting desirable features or classifiers. TSAPA is also capable of predicting tissue-specific poly(A) sites in unannotated intergenic regions. TSAPA will be a valuable addition to the community for studying dynamics of APA in plants. https://github.com/BMILAB/TSAPA. Supplementary data are available at Bioinformatics online.

  7. A dermal HOX transcriptional program regulates site-specific epidermal fate

    PubMed Central

    Rinn, John L.; Wang, Jordon K.; Allen, Nancy; Brugmann, Samantha A.; Mikels, Amanda J.; Liu, Helen; Ridky, Todd W.; Stadler, H. Scott; Nusse, Roel; Helms, Jill A.; Chang, Howard Y.

    2008-01-01

    Reciprocal epithelial–mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration. PMID:18245445

  8. A dermal HOX transcriptional program regulates site-specific epidermal fate.

    PubMed

    Rinn, John L; Wang, Jordon K; Allen, Nancy; Brugmann, Samantha A; Mikels, Amanda J; Liu, Helen; Ridky, Todd W; Stadler, H Scott; Nusse, Roel; Helms, Jill A; Chang, Howard Y

    2008-02-01

    Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

  9. Efficient Site-Specific Labeling of Proteins via Cysteines

    PubMed Central

    Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

    2011-01-01

    Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

  10. Efficient site-specific labeling of proteins via cysteines.

    PubMed

    Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

    2008-03-01

    Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

  11. The λ Integrase Site-specific Recombination Pathway

    PubMed Central

    LANDY, ARTHUR

    2017-01-01

    The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in Mobile DNA II, there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics. PMID:26104711

  12. Temporally-Controlled Site-Specific Recombination in Zebrafish

    PubMed Central

    Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

    2009-01-01

    Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms. PMID:19247481

  13. Minimal Data and Site Specific Approaches

    EPA Science Inventory

    As part of a workshop, Tools for Assessing Stream Dissolved Minerals, approaches and EPA tools are described for site specific development of water quality criteria based on observations from Arkansas streams using minimal data. Discussion topics will include site-specific appro...

  14. Genome-wide detection of conservative site-specific recombination in bacteria

    PubMed Central

    Mathias Garrett, Elizabeth; Camilli, Andrew

    2018-01-01

    The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features. Here, we report an easy-to-implement method based on high-throughput paired-end sequencing for genome-wide detection of conservative site-specific recombination on a single-nucleotide level. We demonstrate the effectiveness of the method by successfully detecting several novel inversion sites in an epidemic isolate of the enteric pathogen Clostridium difficile. Using an experimental approach, we validate the inversion potential of all detected sites in C. difficile and quantify their prevalence during exponential and stationary growth in vitro. In addition, we demonstrate that the master recombinase RecV is responsible for the inversion of some but not all invertible sites. Using a fluorescent gene-reporter system, we show that at least one gene from a two-component system located next to an invertible site is expressed in an on-off mode reminiscent of phase variation. We further demonstrate the applicability of our method by mining 209 publicly available sequencing datasets and show that conservative site-specific recombination is common in the bacterial realm but appears to be absent in some lineages. Finally, we show that the gene content associated with the inversion sites is diverse and goes beyond traditionally described surface components. Overall, our method provides a robust platform for detection of conservative site-specific recombination in bacteria and opens a new avenue for global exploration of this important phenomenon. PMID:29621238

  15. Statistical and Economic Techniques for Site-specific Nematode Management.

    PubMed

    Liu, Zheng; Griffin, Terry; Kirkpatrick, Terrence L

    2014-03-01

    Recent advances in precision agriculture technologies and spatial statistics allow realistic, site-specific estimation of nematode damage to field crops and provide a platform for the site-specific delivery of nematicides within individual fields. This paper reviews the spatial statistical techniques that model correlations among neighboring observations and develop a spatial economic analysis to determine the potential of site-specific nematicide application. The spatial econometric methodology applied in the context of site-specific crop yield response contributes to closing the gap between data analysis and realistic site-specific nematicide recommendations and helps to provide a practical method of site-specifically controlling nematodes.

  16. Characterisation of the site-specific monoPEGylated rhG-CSF analogue pegteograstim.

    PubMed

    Hong, Jeungwoon; Lee, Byoungju; Kang, Kwanyub; Lee, Seung-Hoon; Ryu, Jaehwan; Jung, Gangsoo; Oh, Jaetaek; Jo, Eui-Cheol; Kim, Chan-Wha

    2018-01-01

    We describe the characterisation of a novel monoPEGylated recombinant human granulocyte colony-stimulating factor analogue, pegteograstim (Neulapeg), prepared by site-specific 20 kDa maleimide-PEG conjugation. An additional cysteine was inserted between Gly136 and Ala137 of filgrastim (methionyl human granulocyte colony-stimulating factor) for site-specific PEGylation, and Cys18 of filgrastim was replaced with Ser18 to prevent unwanted PEGylation. Pegteograstim was produced by Escherichia coli and purified by cation exchange chromatography, and its structural, physicochemical, biological and immunological properties were investigated. Male Sprague-Dawley rats were administered pegteograstim (100 μg/kg) and the pharmacokinetics and pharmacodynamics compared with those of filgrastim. The results of long-term stability testing of pegteograstim revealed no significant change in its quality attributes at 2-8 °C for 36 months. In addition, pegteograstim was stable under the accelerated conditions (25 ± 2 °C, RH of 60 ± 5%) for 6 months. The site-specific monoPEGylated pegteograstim is a highly pure, stable and novel drug for long-lasting treatment of chemotherapy-induced neutropenia. Copyright © 2017. Published by Elsevier Ltd.

  17. Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering.

    PubMed

    Karimova, Madina; Splith, Victoria; Karpinski, Janet; Pisabarro, M Teresa; Buchholz, Frank

    2016-07-22

    Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets.

  18. Recruiting Human Microbiome Shotgun Data to Site-Specific Reference Genomes

    PubMed Central

    Xie, Gary; Lo, Chien-Chi; Scholz, Matthew; Chain, Patrick S. G.

    2014-01-01

    The human body consists of innumerable multifaceted environments that predispose colonization by a number of distinct microbial communities, which play fundamental roles in human health and disease. In addition to community surveys and shotgun metagenomes that seek to explore the composition and diversity of these microbiomes, there are significant efforts to sequence reference microbial genomes from many body sites of healthy adults. To illustrate the utility of reference genomes when studying more complex metagenomes, we present a reference-based analysis of sequence reads generated from 55 shotgun metagenomes, selected from 5 major body sites, including 16 sub-sites. Interestingly, between 13% and 92% (62.3% average) of these shotgun reads were aligned to a then-complete list of 2780 reference genomes, including 1583 references for the human microbiome. However, no reference genome was universally found in all body sites. For any given metagenome, the body site-specific reference genomes, derived from the same body site as the sample, accounted for an average of 58.8% of the mapped reads. While different body sites did differ in abundant genera, proximal or symmetrical body sites were found to be most similar to one another. The extent of variation observed, both between individuals sampled within the same microenvironment, or at the same site within the same individual over time, calls into question comparative studies across individuals even if sampled at the same body site. This study illustrates the high utility of reference genomes and the need for further site-specific reference microbial genome sequencing, even within the already well-sampled human microbiome. PMID:24454771

  19. WAG 2 remedial investigation and site investigation site-specific work plan/health and safety checklist for the sediment transport modeling task

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

    Holt, V.L.; Baron, L.A.

    1994-05-01

    This site-specific Work Plan/Health and Safety Checklist (WP/HSC) is a supplement to the general health and safety plan (HASP) for Waste Area Grouping (WAG) 2 remedial investigation and site investigation (WAG 2 RI&SI) activities [Health and Safety Plan for the Remedial Investigation and Site Investigation of Waste Area Grouping 2 at the Oak Ridge National Laboratory, Oak Ridge, Tennessee (ORNL/ER-169)] and provides specific details and requirements for the WAG 2 RI&SI Sediment Transport Modeling Task. This WP/HSC identifies specific site operations, site hazards, and any recommendations by Oak Ridge National Laboratory (ORNL) health and safety organizations [i.e., Industrial Hygiene (IH),more » Health Physics (HP), and/or Industrial Safety] that would contribute to the safe completion of the WAG 2 RI&SI. Together, the general HASP for the WAG 2 RI&SI (ORNL/ER-169) and the completed site-specific WP/HSC meet the health and safety planning requirements specified by 29 CFR 1910.120 and the ORNL Hazardous Waste Operations and Emergency Response (HAZWOPER) Program Manual. In addition to the health and safety information provided in the general HASP for the WAG 2 RI&SI, details concerning the site-specific task are elaborated in this site-specific WP/HSC, and both documents, as well as all pertinent procedures referenced therein, will be reviewed by all field personnel prior to beginning operations.« less

  20. SITE-SPECIFIC CHARACTERIZATION OF SOIL RADON POTENTIALS

    EPA Science Inventory

    The report presents a theoretical basis for measuring site-specific radon potentials. However, the empirical measurements suggest that the precision of such measurements is marginal, leaving an uncertainty of about a factor of 2 in site-specific estimates. Although this may be us...

  1. Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering

    PubMed Central

    Karimova, Madina; Splith, Victoria; Karpinski, Janet; Pisabarro, M. Teresa; Buchholz, Frank

    2016-01-01

    Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets. PMID:27444945

  2. Site-Specific Rules for Risk Assessment.

    ERIC Educational Resources Information Center

    Tucker, William; And Others

    1994-01-01

    This article examines the development of regulatory guidance upon which the technology of risk assessment has matured into a decision-making method of choice. It examines in particular the role of site-specific risk assessment at Superfund sites. (LZ)

  3. The Connectivity Between Site-Specific Life Cycle Impact Assessment and Site-Specific Weighting

    EPA Science Inventory

    The goal of many LCIAs is to come to a single score with all of the impacts from a wide variety of impact assessments weighted to form this single score. My past experiences with developing site-specific impact assessment methodologies and how this can change the valuation porti...

  4. Musite, a tool for global prediction of general and kinase-specific phosphorylation sites.

    PubMed

    Gao, Jianjiong; Thelen, Jay J; Dunker, A Keith; Xu, Dong

    2010-12-01

    (including condition-specific models) from users' own data. In addition, with its easily extensible open source application programming interface, Musite is aimed at being an open platform for community-based development of machine learning-based phosphorylation site prediction applications. Musite is available at http://musite.sourceforge.net/.

  5. Localized melt-scan strategy for site specific control of grain size and primary dendrite arm spacing in electron beam additive manufacturing

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

    Raghavan, Narendran; Simunovic, Srdjan; Dehoff, Ryan

    In addition to design geometry, surface roughness, and solid-state phase transformation, solidification microstructure plays a crucial role in controlling the performance of additively manufactured components. Crystallographic texture, primary dendrite arm spacing (PDAS), and grain size are directly correlated to local solidification conditions. We have developed a new melt-scan strategy for inducing site specific, on-demand control of solidification microstructure. We were able to induce variations in grain size (30 μm–150 μm) and PDAS (4 μm - 10 μm) in Inconel 718 parts produced by the electron beam additive manufacturing system (Arcam®). A conventional raster melt-scan resulted in a grain size ofmore » about 600 μm. The observed variations in grain size with different melt-scan strategies are rationalized using a numerical thermal and solidification model which accounts for the transient curvature of the melt pool and associated thermal gradients and liquid-solid interface velocities. The refinement in grain size at high cooling rates (>104 K/s) is also attributed to the potential heterogeneous nucleation of grains ahead of the epitaxially growing solidification front. The variation in PDAS is rationalized using a coupled numerical-theoretical model as a function of local solidification conditions (thermal gradient and liquid-solid interface velocity) of the melt pool.« less

  6. [3H]aniracetam binds to specific recognition sites in brain membranes.

    PubMed

    Fallarino, F; Genazzani, A A; Silla, S; L'Episcopo, M R; Camici, O; Corazzi, L; Nicoletti, F; Fioretti, M C

    1995-08-01

    [3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Site-Specific Infrared Probes of Proteins

    PubMed Central

    Ma, Jianqiang; Pazos, Ileana M.; Zhang, Wenkai; Culik, Robert M.; Gai, Feng

    2015-01-01

    Infrared spectroscopy has played an instrumental role in studying a wide variety of biological questions. However, in many cases it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and/or environmental information in a site-specific manner. To overcome this limitation, many recent efforts have been dedicated to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural and/or environmental properties. In this Review, we highlight some recent advancements of this rapidly growing research area. PMID:25580624

  8. 14. "SITE WORK, CIVIL, SITE PLAN." Test Area 1120. Specifications ...

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

    14. "SITE WORK, CIVIL, SITE PLAN." Test Area 1-120. Specifications No. OC2-55-72; Drawing No. 60-09-12; sheet 7 of 148; file no. 1320/58, Rev. C. Stamped: RECORD DRAWING - AS CONSTRUCTED. Below stamp: Contract no. 4338 Rev. C, Date: 16 April 1957. - Edwards Air Force Base, Air Force Rocket Propulsion Laboratory, Leuhman Ridge near Highways 58 & 395, Boron, Kern County, CA

  9. Site-specific nutrient management systems

    USDA-ARS?s Scientific Manuscript database

    Site-specific nutrient management systems were created to manage for spatial and temporal variability in biophysical factors that determine the availability and demand of crop nutrients. These systems differ among geographical regions in the information utilized and way they operate to accomplish th...

  10. 78 FR 14088 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act requires that public notice of this meeting be announced in the Federal Register.

  11. Phase Structure and Site Preference Behavior of Ternary Alloying Additions to PdTi and PtTi Shape-Memory Alloys

    NASA Technical Reports Server (NTRS)

    Bozzolo, Guillermo; Mosca, Hugo O.; Noebe, Ronald D.

    2006-01-01

    The phasc structure and concentration dependence of the lattice parameter and energy of formation of ternary Pd-'I-X and Pt-Ti-X alloys for a large number of ternary alloying additions X (X = Na, Mg, Al, Si, Sc. V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Ag, Cd, Hf, Ta, W, Re, Os, Ir) are investigated with an atomistic modeling approach. In addition, a detailed description of the site preference behavior of such additions showing that the elements can be grouped according to their absolute preference for a specific site, regardless of concentration, or preference for available sites in the deficient sublattice is provided.

  12. 75 FR 65310 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-22

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada Test Site. The Federal Advisory... Board is to make recommendations to DOE-EM and site management in the areas of environmental restoration...

  13. Site-Specific Carbon Isotopes in Organics

    NASA Astrophysics Data System (ADS)

    Piasecki, A.; Eiler, J. M.

    2012-12-01

    Natural organic molecules exhibit a wide range of internal site-specific isotope variation (i.e., molecules with same isotopic substitution type but different site). Such variations are generally unconstrained by bulk isotopic measurements. If known, site-specific variations might constrain temperatures of equilibrium, mechanisms of formation or consumption reactions, and possibly other details. For example, lipids can exhibit carbon isotope differences of up to 30‰ between adjacent carbon sites as a result of fractionations arising during decarboxylation of pyruvate and other steps in lipid biosynthesis(1). We present a method for site-specific carbon isotope analysis of propane, based on high-resolution, multi-collector gas source mass spectrometry, using a novel prototype instrument - the Thermo MAT 253 Ultra. This machine has an inlet system and electron bombardment ion source resembling those in conventional stable isotope gas source mass spectrometers, and the energy filter, magnet, and detector array resembling those in multi-collector ICPMS and TIMS. The detector array has 7 detector positions, 6 of which are movable, and each of which can collect ions with either a faraday cup (read through amplifiers ranging from 107-1012 ohms) or an SEM. High mass resolving power (up to 27,000, MRP = M/dM definition) is achieved through a narrow entrance slit, adjustable from 250 to 5 μm. Such resolution can cleanly separate isobaric interferences between isotopologues of organic molecules having the same cardinal mass (e.g., 13CH3 and 12CH2D). We use this technology to analyze the isotopologues and fragments of propane, and use such data to solve for the site-specific carbon isotope fractionation. By measuring isotopologues of both the one-carbon (13CH3) and the two-carbon (13C12CH4) fragment ion, we can solve for both bulk δ13C and the difference in δ13C between the terminal and central carbon position. We tested this method by analyzing mixtures between natural

  14. Site-specific DNA Inversion by Serine Recombinases

    PubMed Central

    2015-01-01

    Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

  15. 77 FR 60688 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  16. 78 FR 26005 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-03

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  17. 77 FR 53193 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-31

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  18. 77 FR 13104 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-05

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  19. 77 FR 39235 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-02

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  20. 78 FR 65979 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  1. 78 FR 716 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  2. 78 FR 54461 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  3. 77 FR 24695 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-25

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. . 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  4. 76 FR 81487 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-28

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  5. Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

    PubMed

    Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

    2015-01-01

    In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for

  6. Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

    PubMed Central

    Jayasena, S D; Johnston, B H

    1992-01-01

    tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

  7. 78 FR 16260 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-14

    ...On March 4, 2013, the Department of Energy (DOE) published a notice of open meeting announcing a meeting on March 25-26, 2013 of the Environmental Management Site-Specific Advisory Board, Savannah River Site (78 FR 14088). This document makes a correction to that notice.

  8. 78 FR 40130 - Environmental Management Site-Specific Advisory Board, Savannah River Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-03

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Savannah River Site. The Federal Advisory Committee Act (Pub. L. No. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  9. 78 FR 59012 - Environmental Management Site-Specific Advisory Board Chairs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-25

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board Chairs AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB) Chairs. The Federal Advisory Committee Act (Pub... and site management in the areas of environmental restoration, waste management, and related...

  10. 77 FR 59598 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-28

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  11. 75 FR 9404 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-02

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  12. 75 FR 61711 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-06

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  13. 75 FR 56526 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-16

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Initiative Workshop of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  14. 75 FR 66074 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-27

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  15. 75 FR 54600 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-08

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  16. 76 FR 5147 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-28

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  17. 75 FR 24686 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-05

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management and...

  18. 77 FR 4027 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-26

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management, and...

  19. 76 FR 80354 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-23

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management, and...

  20. 78 FR 20311 - Environmental Management Site-Specific Advisory Board Chairs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-04

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board Chairs AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB) Chairs. The Federal Advisory Committee Act (Pub... Board is to make recommendations to DOE-EM and site management in the areas of environmental restoration...

  1. 75 FR 82004 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-29

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management, and...

  2. 77 FR 12044 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-28

    ... Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub... Board is to make recommendations to DOE-EM and site management in the areas of environmental restoration... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY...

  3. 76 FR 21878 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-19

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... Board: The purpose of the Board is to make recommendations to DOE-EM and site management in the areas of...

  4. 75 FR 65615 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-26

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... the Board: The purpose of the Board is to make recommendations to DOE-EM and site management in the...

  5. 75 FR 82003 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-29

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... the Board: The purpose of the Board is to make recommendations to DOE-EM and site management in the...

  6. 77 FR 75626 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-21

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act...: Purpose of the Board: The purpose of the Board is to make recommendations to DOE-EM and site management in...

  7. 77 FR 37390 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-21

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... Board: The purpose of the Board is to make recommendations to DOE-EM and site management in the areas of...

  8. 77 FR 2283 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-17

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... Board: The purpose of the Board is to make recommendations to DOE-EM and site management in the areas of...

  9. 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.

  10. 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

  11. Site- and species-specific hydrolysis rates of heroin.

    PubMed

    Szöcs, Levente; Orgován, Gábor; Tóth, Gergő; Kraszni, Márta; Gergó, Lajos; Hosztafi, Sándor; Noszál, Béla

    2016-06-30

    The hydroxide-catalyzed non-enzymatic, simultaneous and consecutive hydrolyses of diacetylmorphine (DAM, heroin) are quantified in terms of 10 site- and species-specific rate constants in connection with also 10 site- and species-specific acid-base equilibrium constants, comprising all the 12 coexisting species in solution. This characterization involves the major and minor decomposition pathways via 6-acetylmorphine and 3-acetylmorphine, respectively, and morphine, the final product. Hydrolysis has been found to be 18-120 times faster at site 3 than at site 6, depending on the status of the amino group and the rest of the molecule. Nitrogen protonation accelerates the hydrolysis 5-6 times at site 3 and slightly less at site 6. Hydrolysis rate constants are interpreted in terms of intramolecular inductive effects and the concomitant local electron densities. Hydrolysis fraction, a new physico-chemical parameter is introduced and determined to quantify the contribution of the individual microspecies to the overall hydrolysis. Hydrolysis fractions are depicted as a function of pH. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. 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

  13. Site-specific genetic recombination: hops, flips, and flops.

    PubMed

    Sadowski, P D

    1993-06-01

    Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans. Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements. This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

  14. Breeding site selection by coho salmon (Oncorhynchus kisutch) in relation to large wood additions and factors that influence reproductive success

    USGS Publications Warehouse

    Clark, Steven M.; Dunham, Jason B.; McEnroe, Jeffery R.; Lightcap, Scott W.

    2014-01-01

    The fitness of female Pacific salmon (Oncorhynchus spp.) with respect to breeding behavior can be partitioned into at least four fitness components: survival to reproduction, competition for breeding sites, success of egg incubation, and suitability of the local environment near breeding sites for early rearing of juveniles. We evaluated the relative influences of habitat features linked to these fitness components with respect to selection of breeding sites by coho salmon (Oncorhynchus kisutch). We also evaluated associations between breeding site selection and additions of large wood, as the latter were introduced into the study system as a means of restoring habitat conditions to benefit coho salmon. We used a model selection approach to organize specific habitat features into groupings reflecting fitness components and influences of large wood. Results of this work suggest that female coho salmon likely select breeding sites based on a wide range of habitat features linked to all four hypothesized fitness components. More specifically, model parameter estimates indicated that breeding site selection was most strongly influenced by proximity to pool-tail crests and deeper water (mean and maximum depths). Linkages between large wood and breeding site selection were less clear. Overall, our findings suggest that breeding site selection by coho salmon is influenced by a suite of fitness components in addition to the egg incubation environment, which has been the emphasis of much work in the past.

  15. Inducible model for β-six-mediated site-specific recombination in mammalian cells

    PubMed Central

    Servert, Pilar; Garcia-Castro, Javier; Díaz, Vicente; Lucas, Daniel; Gonzalez, Manuel A.; Martínez-A, Carlos; Bernad, Antonio

    2006-01-01

    The prokaryotic β recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in chromatin-integrated substrates. Here, we demonstrate that an enhanced green fluorescent protein (EGFP)-fused version of β recombinase (β-EGFP) is fully active, retaining most specific activity. It is used to develop a recombination-dependent activatable gene expression (RAGE) system based on the androgen receptor (AR) ligand-binding domain (LBD). Two hybrid molecules, a direct fusion of the LBD-AR to the C-terminus of β recombinase (β-AR) and a triple fusion of β-EGFP to the same ligand-binding domain (β-EGFP-AR), were engineered and their subcellular behavior, stability and catalytic activity were evaluated. Both chimeric β recombinase proteins showed in vivo inducible recombinogenic activity dependent on addition of an androgen receptor agonist, although the β-AR fusion protein demonstrated more accurate ligand-dependent translocation from cytoplasm to nucleus. PMID:16394020

  16. Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.

    PubMed

    Yang, Jing; Tallman, Keri A; Porter, Ned A; Liebler, Daniel C

    2015-03-03

    Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner.

  17. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

    PubMed

    Kumar, Raj; Calhoun, William J

    2008-12-01

    Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and

  18. Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure.

    PubMed

    Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

    2014-04-08

    Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a "site-specific" homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional "non-site-specific" allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view.

  19. 76 FR 55370 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-07

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY...-Wide Environmental Impact Statement (EIS) Committee of the Environmental Management Site- Specific... the areas of environmental restoration, waste management, and related activities. Purpose of the...

  20. SITE SPECIFIC REFERENCE PERSON PARAMETERS AND DERIVED CONCENTRATION STANDARDS FOR THE SAVANNAH RIVER SITE

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

    Jannik, T.

    2013-03-14

    The purpose of this report is twofold. The first is to develop a set of behavioral parameters for a reference person specific for the Savannah River Site (SRS) such that the parameters can be used to determine dose to members of the public in compliance with Department of Energy (DOE) Order 458.1 “Radiation Protection of the Public and the Environment.” A reference person is a hypothetical, gender and age aggregation of human physical and physiological characteristics arrived at by international consensus for the purpose of standardizing radiation dose calculations. DOE O 458.1 states that compliance with the annual dose limitmore » of 100 mrem (1 mSv) to a member of the public may be demonstrated by calculating the dose to the maximally exposed individual (MEI) or to a representative person. Historically, for dose compliance, SRS has used the MEI concept, which uses adult dose coefficients and adult male usage parameters. Beginning with the 2012 annual site environmental report, SRS will be using the representative person concept for dose compliance. The dose to a representative person will be based on 1) the SRS-specific reference person usage parameters at the 95th percentile of appropriate national or regional data, which are documented in this report, 2) the reference person (gender and age averaged) ingestion and inhalation dose coefficients provided in DOE Derived Concentration Technical Standard (DOE-STD-1196-2011), and 3) the external dose coefficients provided in the DC_PAK3 toolbox. The second purpose of this report is to develop SRS-specific derived concentration standards (DCSs) for all applicable food ingestion pathways, ground shine, and water submersion. The DCS is the concentration of a particular radionuclide in water, in air, or on the ground that results in a member of the public receiving 100 mrem (1 mSv) effective dose following continuous exposure for one year. In DOE-STD-1196-2011, DCSs were developed for the ingestion of water

  1. 76 FR 57981 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-19

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... environmental restoration, waste management and related activities. Tentative Agenda Call to Order...

  2. 77 FR 29997 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-21

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... environmental restoration, waste management and related activities. Tentative Agenda Call to Order...

  3. 76 FR 50204 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-12

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Nevada AGENCY...-Wide Environmental Impact Statement (EIS) Committee of the Environmental Management Site- Specific... management in the areas of environmental restoration, waste management, and related activities. Purpose of...

  4. 76 FR 78909 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-20

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... the areas of environmental restoration, waste management, and related activities. Tentative Agenda...

  5. 76 FR 36100 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-21

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... environmental restoration, waste management and related activities. Tentative Agenda Call to Order...

  6. Environmental projects, volume 11. Environmental assessment: Addition to operations building, Mars site

    NASA Technical Reports Server (NTRS)

    1990-01-01

    An Environmental Assessment was performed of the proposed addition to building G-86 at the Mars Site, which will provide space for new electronic equipment to consolidate the Deep Space Network (DSN) support facilities from other Goldstone Deep Space Communication Complex (GDSCC) sites at the Mars Site, and will include a fifth telemetry and command group with its associated link monitor, control processor, and operator consoles. The addition of these facilities will increase the capability of the DSN to support future sophisticated NASA spacecraft missions such as the International Solar and Terrestrial Physics (ISTP) Program. The planned construction of this building addition requires an Environmental Assessment (EA) document that records the existing environmental conditions at the Mars Site, that analyzes the environmental effects that possibly could be expected from the construction and use of the new building addition, and that recommends measures to be taken to mitigate any possible deleterious environmental effects.

  7. 77 FR 51789 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... management and related activities. Tentative Agenda Call to Order, Introductions, Review of Agenda... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act...

  8. 75 FR 19379 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-14

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... areas of environmental restoration, waste management and related activities. Tentative Agenda Call to...

  9. 75 FR 7577 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-22

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act... areas of environmental restoration, waste management and related activities. Tentative Agenda: Call to...

  10. 76 FR 17118 - Environmental Management Site-Specific Advisory Board Chairs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-28

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board Chairs AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB) Chairs. The Federal Advisory Committee Act (Pub... areas of environmental restoration, waste management, and related activities. Tentative Agenda Topics...

  11. 76 FR 62054 - Environmental Management Site-Specific Advisory Board Chairs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-06

    ... environmental restoration, waste management, and related activities. Tentative Agenda Topics [cir] EM Program... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board Chairs AGENCY... of the Environmental Management Site-Specific Advisory Board (EM SSAB) Chairs. The Federal Advisory...

  12. SUPPORTING THE REDEVELOPMENT OF BROWNFIELD SITES USING SITE-SPECIFIC MANAGEMENT APPROACHES AND REDEVELOPMENT TOOLS (SMART)

    EPA Science Inventory

    The Site-Specific Management Approaches and Redevelopment Tools (SMART) provides potential solutions for facilitating the redevelopment of brownfield sites. The term "brownfield site" refers to previously developed property whose reuse may be complicated by the presence of hazar...

  13. Expanding the scope of site-specific recombinases for genetic and metabolic engineering.

    PubMed

    Gaj, Thomas; Sirk, Shannon J; Barbas, Carlos F

    2014-01-01

    Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. © 2013 Wiley Periodicals, Inc.

  14. Expanding the Scope of Site-Specific Recombinases for Genetic and Metabolic Engineering

    PubMed Central

    Gaj, Thomas; Sirk, Shannon J.; Barbas, Carlos F.

    2014-01-01

    Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. PMID:23982993

  15. 77 FR 55813 - Environmental Management Site-Specific Advisory Board Chairs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-11

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board Chairs AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB) Chairs. The Federal Advisory Committee Act (Pub... environmental restoration, waste management, and related activities. Tentative Agenda Topics Tuesday, October 2...

  16. 75 FR 64718 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-20

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Hanford AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act.... ADDRESSES: Red Lion Hanford House, 802 George Washington Way, Richland, Washington. FOR FURTHER INFORMATION...

  17. 75 FR 8051 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-23

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Hanford AGENCY... the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford (known locally as the Hanford Advisory Board [HAB]), River and Plateau, Tank Waste, Public Involvement, Health Safety and...

  18. 76 FR 4645 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-26

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Hanford AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act.... ADDRESSES: Red Lion Hanford House, 802 George Washington Way, Richland, Washington 99352. FOR FURTHER...

  19. Evaluation of site-specific tactics using bifenazate and Neoseiulus californicus for management of Tetranychus urticae (Acari: Tetranychidae) in strawberries.

    PubMed

    Liu, Ruohan; Nyoike, Teresia W; Liburd, Oscar E

    2016-10-01

    Greenhouse and field experiments were conducted to evaluate the effectiveness of site-specific tactics for management of the twospotted spider mite, Tetranychus urticae Koch, a major pest of greenhouse and field-grown strawberries (Fragaria x ananassa Duchesne). Two site-specific (spot) treatments, the miticide bifenazate (Acramite(®)) and the predatory mite Neoseiulus californicus McGregor, were compared with whole-plot treatments of bifenazate or N. californicus to determine whether T. urticae could be effectively managed in field-grown strawberry using only site-specific tactics. Additionally, the cost of site-specific tactics was compared with whole-plot treatments to determine the economic value of using site-specific management tactics for T. urticae in strawberries. In the greenhouse, all treatments equivalently reduced the number of T. urticae below control. In the field during the 2011-2012 season, more T. urticae eggs and motiles were in the whole-plot treatments of both N. californicus and bifenazate in the mid-season and late season, respectively, compared with the spot treatments. With the exception of site-specific N. californicus during the 2011-2012 field season, there were no differences in marketable yields between plots with site-specific treatments and whole-plot management. An economic analysis demonstrated a significant cost savings (75.3 %) with site-specific treatments of N. californicus compared with whole-plot application of N. californicus. Similarly, a 24.7 % reduction in cost was achieved in using site-specific bifenazate compared with whole-plot application of bifenazate. The findings indicate that site-specific treatments with N. californicus and bifenazate are competitive alternatives to whole-field application for T. urticae management in strawberries.

  20. Site Specific Management of Cotton Production in the United States

    USDA-ARS?s Scientific Manuscript database

    Site-specific management or precision agriculture, as it is evolving in large-scale crop production, offers promising new methods for managing cotton production for optimized yields, maximized profitability, and minimized environmental pollution. However, adaptation of site-specific theory and meth...

  1. 77 FR 11516 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-27

    ... materials; excess facilities; future land use and long-term stewardship; risk assessment and management; and... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act...

  2. 77 FR 2282 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-17

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Paducah AGENCY... the Environmental Management Site-Specific Advisory Board, Paducah. This notice announces the... scheduling conflicts by members. The next regular meeting will be held on February 16, 2012. DATES: The...

  3. 75 FR 8050 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-23

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Hanford AGENCY... Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act...: The meeting is open to the public. The EM SSAB, Hanford, welcomes the attendance of the public at its...

  4. 76 FR 20651 - Environmental Management Site-Specific Advisory Board Chairs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-13

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board Chairs AGENCY... a meeting on April 13-14, 2011 of the Environmental Management Site-Specific Advisory Board Chairs... R. Butler, Acting Deputy Committee Management Officer. [FR Doc. 2011-8970 Filed 4-8-11; 4:15 pm...

  5. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

    USDA-ARS?s Scientific Manuscript database

    A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they h...

  6. Tree-, stand- and site-specific controls on landscape-scale patterns of transpiration

    NASA Astrophysics Data System (ADS)

    Kathrin Hassler, Sibylle; Weiler, Markus; Blume, Theresa

    2018-01-01

    Transpiration is a key process in the hydrological cycle, and a sound understanding and quantification of transpiration and its spatial variability is essential for management decisions as well as for improving the parameterisation and evaluation of hydrological and soil-vegetation-atmosphere transfer models. For individual trees, transpiration is commonly estimated by measuring sap flow. Besides evaporative demand and water availability, tree-specific characteristics such as species, size or social status control sap flow amounts of individual trees. Within forest stands, properties such as species composition, basal area or stand density additionally affect sap flow, for example via competition mechanisms. Finally, sap flow patterns might also be influenced by landscape-scale characteristics such as geology and soils, slope position or aspect because they affect water and energy availability; however, little is known about the dynamic interplay of these controls.We studied the relative importance of various tree-, stand- and site-specific characteristics with multiple linear regression models to explain the variability of sap velocity measurements in 61 beech and oak trees, located at 24 sites across a 290 km2 catchment in Luxembourg. For each of 132 consecutive days of the growing season of 2014 we modelled the daily sap velocity and derived sap flow patterns of these 61 trees, and we determined the importance of the different controls.Results indicate that a combination of mainly tree- and site-specific factors controls sap velocity patterns in the landscape, namely tree species, tree diameter, geology and aspect. For sap flow we included only the stand- and site-specific predictors in the models to ensure variable independence. Of those, geology and aspect were most important. Compared to these predictors, spatial variability of atmospheric demand and soil moisture explains only a small fraction of the variability in the daily datasets. However, the temporal

  7. Accounting for both local aquatic community composition and bioavailability in setting site-specific quality standards for zinc.

    PubMed

    Peters, Adam; Simpson, Peter; Moccia, Alessandra

    2014-01-01

    Recent years have seen considerable improvement in water quality standards (QS) for metals by taking account of the effect of local water chemistry conditions on their bioavailability. We describe preliminary efforts to further refine water quality standards, by taking account of the composition of the local ecological community (the ultimate protection objective) in addition to bioavailability. Relevance of QS to the local ecological community is critical as it is important to minimise instances where quality classification using QS does not reconcile with a quality classification based on an assessment of the composition of the local ecology (e.g. using benthic macroinvertebrate quality assessment metrics such as River InVertebrate Prediction and Classification System (RIVPACS)), particularly where ecology is assessed to be at good or better status, whilst chemical quality is determined to be failing relevant standards. The alternative approach outlined here describes a method to derive a site-specific species sensitivity distribution (SSD) based on the ecological community which is expected to be present at the site in the absence of anthropogenic pressures (reference conditions). The method combines a conventional laboratory ecotoxicity dataset normalised for bioavailability with field measurements of the response of benthic macroinvertebrate abundance to chemical exposure. Site-specific QSref are then derived from the 5%ile of this SSD. Using this method, site QSref have been derived for zinc in an area impacted by historic mining activities. Application of QSref can result in greater agreement between chemical and ecological metrics of environmental quality compared with the use of either conventional (QScon) or bioavailability-based QS (QSbio). In addition to zinc, the approach is likely to be applicable to other metals and possibly other types of chemical stressors (e.g. pesticides). However, the methodology for deriving site-specific targets requires

  8. Dancing in Place: Site-Specific Work

    ERIC Educational Resources Information Center

    Metal-Corbin, Josie

    2012-01-01

    In her lecture the 2012 NDA Scholar/Artist, Josie Metal-Corbin, chronicles four decades of working with artists, educators, librarians, and scientists. The kinetic language of dance and the visual impact of specific environments provide provocative opportunities for collaboration, wherein the site becomes the framework or map for the dance design.…

  9. Cre recombinase-mediated site-specific recombination between plant chromosomes.

    PubMed Central

    Qin, M; Bayley, C; Stockton, T; Ow, D W

    1994-01-01

    We report the use of the bacteriophage P1 Cre-lox system for generating conservative site-specific recombination between tobacco chromosomes. Two constructs, one containing a promoterless hygromycin-resistance gene preceded by a lox site (lox-hpt) and the other containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre coding region (35S-lox-cre), were introduced separately into tobacco plants. Crosses between plants harboring either construct produced plants with the two constructs situated on different chromosomes. Plants with recombination events were identified by selecting for hygromycin resistance, a phenotype expressed upon recombination. Molecular analysis showed that these recombination events occurred specifically at the lox sites and resulted in the reciprocal exchange of flanking host DNA. Progenies of these plants showed 67-100% cotransmission of the new transgenes, 35S-lox-hpt and lox-cre, consistent with the preferential cosegregation of translocated chromosomes. These results illustrate that site-specific recombination systems can be useful tools for the large-scale manipulation of eukaryotic chromosomes in vivo. Images PMID:8127869

  10. Engineering peptide ligase specificity by proteomic identification of ligation sites.

    PubMed

    Weeks, Amy M; Wells, James A

    2018-01-01

    Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

  11. Improving Site-Specific Radiological Performance Assessments - 13431

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

    Tauxe, John; Black, Paul; Catlett, Kate

    2013-07-01

    An improved approach is presented for conducting complete and defensible radiological site-specific performance assessments (PAs) to support radioactive waste disposal decisions. The basic tenets of PA were initiated some thirty years ago, focusing on geologic disposals and evaluating compliance with regulations. Some of these regulations were inherently probabilistic (i.e., addressing uncertainty in a quantitative fashion), such as the containment requirements of the U.S. Environmental Protection Agency's (EPA's) 40 CFR 191, Environmental Radiation Protection Standards for Management and Disposal of Spent Nuclear Fuel, High-Level and Transuranic Radioactive Wastes, Chap. 191.13 [1]. Methods of analysis were developed to meet those requirements, butmore » at their core early PAs used 'conservative' parameter values and modeling approaches. This limited the utility of such PAs to compliance evaluation, and did little to inform decisions about optimizing disposal, closure and long-term monitoring and maintenance, or, in general, maintaining doses 'as low as reasonably achievable' (ALARA). This basic approach to PA development in the United States was employed essentially unchanged through the end of the 20. century, principally by the U.S. Department of Energy (DOE). Performance assessments developed in support of private radioactive waste disposal operations, regulated by the U.S. Nuclear Regulatory Commission (NRC) and its agreement states, were typically not as sophisticated. Discussion of new approaches to PA is timely, since at the time of this writing, the DOE is in the midst of revising its Order 435.1, Radioactive Waste Management [2], and the NRC is revising 10 CFR 61, Licensing Requirements for Land Disposal of Radioactive Waste [3]. Over the previous decade, theoretical developments and improved computational technology have provided the foundation for integrating decision analysis (DA) concepts and objective-focused thinking, plus a Bayesian approach to

  12. Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

    PubMed

    Chowdhury, Sandipan; Chanda, Baron

    2010-11-02

    Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

  13. Theory on the mechanism of site-specific DNA-protein interactions in the presence of traps

    NASA Astrophysics Data System (ADS)

    Niranjani, G.; Murugan, R.

    2016-08-01

    The speed of site-specific binding of transcription factor (TFs) proteins with genomic DNA seems to be strongly retarded by the randomly occurring sequence traps. Traps are those DNA sequences sharing significant similarity with the original specific binding sites (SBSs). It is an intriguing question how the naturally occurring TFs and their SBSs are designed to manage the retarding effects of such randomly occurring traps. We develop a simple random walk model on the site-specific binding of TFs with genomic DNA in the presence of sequence traps. Our dynamical model predicts that (a) the retarding effects of traps will be minimum when the traps are arranged around the SBS such that there is a negative correlation between the binding strength of TFs with traps and the distance of traps from the SBS and (b) the retarding effects of sequence traps can be appeased by the condensed conformational state of DNA. Our computational analysis results on the distribution of sequence traps around the putative binding sites of various TFs in mouse and human genome clearly agree well the theoretical predictions. We propose that the distribution of traps can be used as an additional metric to efficiently identify the SBSs of TFs on genomic DNA.

  14. Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

    PubMed Central

    Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

    2017-01-01

    Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

  15. Surface-specific additive manufacturing test artefacts

    NASA Astrophysics Data System (ADS)

    Townsend, Andrew; Racasan, Radu; Blunt, Liam

    2018-06-01

    Many test artefact designs have been proposed for use with additive manufacturing (AM) systems. These test artefacts have primarily been designed for the evaluation of AM form and dimensional performance. A series of surface-specific measurement test artefacts designed for use in the verification of AM manufacturing processes are proposed here. Surface-specific test artefacts can be made more compact because they do not require the large dimensions needed for accurate dimensional and form measurements. The series of three test artefacts are designed to provide comprehensive information pertaining to the manufactured surface. Measurement possibilities include deviation analysis, surface texture parameter data generation, sub-surface analysis, layer step analysis and build resolution comparison. The test artefacts are designed to provide easy access for measurement using conventional surface measurement techniques, for example, focus variation microscopy, stylus profilometry, confocal microscopy and scanning electron microscopy. Additionally, the test artefacts may be simply visually inspected as a comparative tool, giving a fast indication of process variation between builds. The three test artefacts are small enough to be included in every build and include built-in manufacturing traceability information, making them a convenient physical record of the build.

  16. Handball Practice Enhances Bone Mass in Specific Sites Among Prepubescent Boys.

    PubMed

    Missawi, Kawther; Zouch, Mohamed; Chakroun, Yosra; Chaari, Hamada; Tabka, Zouhair; Bouajina, Elyès

    2016-01-01

    This investigation's purpose is to focus on the effects of practicing handball for at least 2 yr on bone acquisition among prepubescent boys. One hundred prepubescent boys aged 10.68 ± 0.85 yr were divided into 2 groups: 50 handball players (HP group) and 50 controls (C group). Bone mineral density (BMD), bone mineral content (BMC), and bone area (BA) were evaluated by using dual-photon X-ray absorptiometry on the whole body, lumbar spine (L2-L4), legs, arms, femoral necks, hips and radiuses. Results showed greater values of BMD in both right and left femoral neck and total hip in handball players than in controls. In addition, handball players had higher values of legs and right total hip BMC than controls without any obvious variation of BA measurement in all sites between groups. All results of the paired t-test displayed an obviously marked variation of bone mass parameters between the left and right sides in the trained group without any marked variation among controls. Data showed an increased BMD of the supporting sites between the left and the right leg among handball players. However, "BMC" results exhibited higher values in the right than in the left total hip, and in the right total radius than in the left correspondent site. In addition, differences in the "BA" measurements were observed in the left total hip and in the right arm. Specific bone sites are markedly stimulated by handball training in prepubescent boys. Copyright © 2016 International Society for Clinical Densitometry. Published by Elsevier Inc. All rights reserved.

  17. 76 FR 39080 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-05

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management, and...

  18. Tree-, stand- and site-specific controls on landscape-scale patterns of transpiration

    NASA Astrophysics Data System (ADS)

    Hassler, Sibylle; Markus, Weiler; Theresa, Blume

    2017-04-01

    Transpiration is a key process in the hydrological cycle and a sound understanding and quantification of transpiration and its spatial variability is essential for management decisions as well as for improving the parameterisation of hydrological and soil-vegetation-atmosphere transfer models. For individual trees, transpiration is commonly estimated by measuring sap flow. Besides evaporative demand and water availability, tree-specific characteristics such as species, size or social status control sap flow amounts of individual trees. Within forest stands, properties such as species composition, basal area or stand density additionally affect sap flow, for example via competition mechanisms. Finally, sap flow patterns might also be influenced by landscape-scale characteristics such as geology, slope position or aspect because they affect water and energy availability; however, little is known about the dynamic interplay of these controls. We studied the relative importance of various tree-, stand- and site-specific characteristics with multiple linear regression models to explain the variability of sap velocity measurements in 61 beech and oak trees, located at 24 sites spread over a 290 km2-catchment in Luxembourg. For each of 132 consecutive days of the growing season of 2014 we modelled the daily sap velocities of these 61 trees and determined the importance of the different predictors. Results indicate that a combination of tree-, stand- and site-specific factors controls sap velocity patterns in the landscape, namely tree species, tree diameter, the stand density, geology and aspect. Compared to these predictors, spatial variability of atmospheric demand and soil moisture explains only a small fraction of the variability in the daily datasets. However, the temporal dynamics of the explanatory power of the tree-specific characteristics, especially species, are correlated to the temporal dynamics of potential evaporation. Thus, transpiration estimates at the

  19. Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

    DTIC Science & Technology

    2010-06-01

    SITE-SPECIFIC DIFFERENTIATION OF FIBROBLASTS IN NORMAL AND SCLERODERMA SKIN PRINCIPAL INVESTIGATOR: Howard Y. Chang, M.D., Ph.D...2010 4. TITLE AND SUBTITLE Site-Specific Differentiation of Fibroblasts in Normal and 5a. CONTRACT NUMBER Scleroderma Skin 5b. GRANT NUMBER...activated fibroblasts from SSc. 15. SUBJECT TERMS Scleroderma , fibroblasts, gene expression 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF

  20. 20. Photographic copy of an asconstructed site plan for additions ...

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

    20. Photographic copy of an as-constructed site plan for additions to North Base: Job No. A(8-1), Military Construction, Materiel Command Flight Test Base, Muroc, California; Additional Construction, Location Plan, Sheet No. 2, October 1943. Reproduced from the holdings of the National Archives, Pacific Southwest Region - Edwards Air Force Base, North Base, North Base Road, Boron, Kern County, CA

  1. Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

    PubMed Central

    Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

    1999-01-01

    The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

  2. Proteome-wide Light/Dark Modulation of Thiol Oxidation in Cyanobacteria Revealed by Quantitative Site-specific Redox Proteomics*

    PubMed Central

    Guo, Jia; Nguyen, Amelia Y.; Dai, Ziyu; Su, Dian; Gaffrey, Matthew J.; Moore, Ronald J.; Jacobs, Jon M.; Monroe, Matthew E.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.; Qian, Wei-Jun

    2014-01-01

    Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of ∼2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for ∼1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover, the redox sensitivity along with the stoichiometric data enabled prediction of potential functional Cys-sites for proteins of interest. The functional significance of redox-sensitive Cys-sites in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin (AhpC/TSA family protein Sll1621), and glucose 6-phosphate dehydrogenase was further confirmed with site-specific mutagenesis and biochemical studies. Together, our findings provide significant insights into the broad redox regulation of photosynthetic organisms. PMID:25118246

  3. Extensively Parameterized Mutation-Selection Models Reliably Capture Site-Specific Selective Constraint.

    PubMed

    Spielman, Stephanie J; Wilke, Claus O

    2016-11-01

    The mutation-selection model of coding sequence evolution has received renewed attention for its use in estimating site-specific amino acid propensities and selection coefficient distributions. Two computationally tractable mutation-selection inference frameworks have been introduced: One framework employs a fixed-effects, highly parameterized maximum likelihood approach, whereas the other employs a random-effects Bayesian Dirichlet Process approach. While both implementations follow the same model, they appear to make distinct predictions about the distribution of selection coefficients. The fixed-effects framework estimates a large proportion of highly deleterious substitutions, whereas the random-effects framework estimates that all substitutions are either nearly neutral or weakly deleterious. It remains unknown, however, how accurately each method infers evolutionary constraints at individual sites. Indeed, selection coefficient distributions pool all site-specific inferences, thereby obscuring a precise assessment of site-specific estimates. Therefore, in this study, we use a simulation-based strategy to determine how accurately each approach recapitulates the selective constraint at individual sites. We find that the fixed-effects approach, despite its extensive parameterization, consistently and accurately estimates site-specific evolutionary constraint. By contrast, the random-effects Bayesian approach systematically underestimates the strength of natural selection, particularly for slowly evolving sites. We also find that, despite the strong differences between their inferred selection coefficient distributions, the fixed- and random-effects approaches yield surprisingly similar inferences of site-specific selective constraint. We conclude that the fixed-effects mutation-selection framework provides the more reliable software platform for model application and future development. © The Author 2016. Published by Oxford University Press on behalf of the

  4. 40 CFR 228.6 - Specific criteria for site selection.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    .... 228.6 Section 228.6 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING CRITERIA FOR THE MANAGEMENT OF DISPOSAL SITES FOR OCEAN DUMPING § 228.6 Specific criteria for site... of special scientific importance and other legitimate uses of the ocean; (9) The existing water...

  5. DNA-binding regulates site-specific ubiquitination of IRF-1.

    PubMed

    Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

    2013-02-01

    Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

  6. 18. Photographic copy of site plan for additions to North ...

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

    18. Photographic copy of site plan for additions to North Base: Job No. Muroc A(511), Military Construction, Third District Region, San Bernardino, California; Muroc Bombing Range, Muroc Lake, Calif; Additional Temporary Construction, Materiel Center Flight Test Base, Location Plan, February 1943. Reproduced from the holdings of the National Archives, Pacific Southwest Region - Edwards Air Force Base, North Base, North Base Road, Boron, Kern County, CA

  7. GPS-PAIL: prediction of lysine acetyltransferase-specific modification sites from protein sequences.

    PubMed

    Deng, Wankun; Wang, Chenwei; Zhang, Ying; Xu, Yang; Zhang, Shuang; Liu, Zexian; Xue, Yu

    2016-12-22

    Protein acetylation catalyzed by specific histone acetyltransferases (HATs) is an essential post-translational modification (PTM) and involved in the regulation a broad spectrum of biological processes in eukaryotes. Although several ten thousands of acetylation sites have been experimentally identified, the upstream HATs for most of the sites are unclear. Thus, the identification of HAT-specific acetylation sites is fundamental for understanding the regulatory mechanisms of protein acetylation. In this work, we first collected 702 known HAT-specific acetylation sites of 205 proteins from the literature and public data resources, and a motif-based analysis demonstrated that different types of HATs exhibit similar but considerably distinct sequence preferences for substrate recognition. Using 544 human HAT-specific sites for training, we constructed a highly useful tool of GPS-PAIL for the prediction of HAT-specific sites for up to seven HATs, including CREBBP, EP300, HAT1, KAT2A, KAT2B, KAT5 and KAT8. The prediction accuracy of GPS-PAIL was critically evaluated, with a satisfying performance. Using GPS-PAIL, we also performed a large-scale prediction of potential HATs for known acetylation sites identified from high-throughput experiments in nine eukaryotes. Both online service and local packages were implemented, and GPS-PAIL is freely available at: http://pail.biocuckoo.org.

  8. GPS-PAIL: prediction of lysine acetyltransferase-specific modification sites from protein sequences

    PubMed Central

    Deng, Wankun; Wang, Chenwei; Zhang, Ying; Xu, Yang; Zhang, Shuang; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein acetylation catalyzed by specific histone acetyltransferases (HATs) is an essential post-translational modification (PTM) and involved in the regulation a broad spectrum of biological processes in eukaryotes. Although several ten thousands of acetylation sites have been experimentally identified, the upstream HATs for most of the sites are unclear. Thus, the identification of HAT-specific acetylation sites is fundamental for understanding the regulatory mechanisms of protein acetylation. In this work, we first collected 702 known HAT-specific acetylation sites of 205 proteins from the literature and public data resources, and a motif-based analysis demonstrated that different types of HATs exhibit similar but considerably distinct sequence preferences for substrate recognition. Using 544 human HAT-specific sites for training, we constructed a highly useful tool of GPS-PAIL for the prediction of HAT-specific sites for up to seven HATs, including CREBBP, EP300, HAT1, KAT2A, KAT2B, KAT5 and KAT8. The prediction accuracy of GPS-PAIL was critically evaluated, with a satisfying performance. Using GPS-PAIL, we also performed a large-scale prediction of potential HATs for known acetylation sites identified from high-throughput experiments in nine eukaryotes. Both online service and local packages were implemented, and GPS-PAIL is freely available at: http://pail.biocuckoo.org. PMID:28004786

  9. 75 FR 64718 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-20

    ... Industrial Sites and Soils Committees of the Environmental Management Site-Specific Advisory Board (EM SSAB... Test Site including decontamination, closure, re-use and/or demolition. Purpose of the Soils Committee: The purpose of the Committee is to focus on issues related to soil contamination at the Nevada Test...

  10. 40 CFR 228.6 - Specific criteria for site selection.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Specific criteria for site selection. 228.6 Section 228.6 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN... designation promulgation as an environmental assessment of the impact of the use of the site for disposal, and...

  11. A grammar inference approach for predicting kinase specific phosphorylation sites.

    PubMed

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

  12. 75 FR 71677 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

    ... Industrial Sites and Soils Committees of the Environmental Management Site-Specific Advisory Board (EM SSAB... the Soils Committee: The purpose of the Committee is to focus on issues related to soil contamination...

  13. 76 FR 5365 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-31

    ... Industrial Sites and Soils Committees of the Environmental Management Site-Specific Advisory Board (EM SSAB.... Purpose of the Soils Committee: The purpose of the Committee is to focus on issues related to soil...

  14. A Modular Vaccine Development Platform Based on Sortase-Mediated Site-Specific Tagging of Antigens onto Virus-Like Particles

    PubMed Central

    Tang, Shubing; Xuan, Baoqin; Ye, Xiaohua; Huang, Zhong; Qian, Zhikang

    2016-01-01

    Virus-like particles (VLPs) can be used as powerful nanoscale weapons to fight against virus infection. In addition to direct use as vaccines, VLPs have been extensively exploited as platforms on which to display foreign antigens for prophylactic vaccination and immunotherapeutic treatment. Unfortunately, fabrication of new chimeric VLP vaccines in a versatile, site-specific and highly efficient manner is beyond the capability of traditional VLP vaccine design approaches, genetic insertion and chemical conjugation. In this study, we described a greatly improved VLP display strategy by chemoenzymatic site-specific tailoring antigens on VLPs surface with high efficiency. Through the transpeptidation mediated by sortase A, one protein and two epitopes containing N-terminal oligoglycine were conjugated to the LPET motif on the surface of hepatitis B virus core protein (HBc) VLPs with high density. All of the new chimeric VLPs induced strong specific IgG responses. Furthermore, the chimeric VLPs with sortase A tagged enterovirus 71 (EV71) SP70 epitope could elicit effective antibodies against EV71 lethal challenging as well as the genetic insertion chimeric VLPs. The sortase A mediated chemoenzymatic site-specific tailoring of the HBc VLP approach shows great potential in new VLP vaccine design for its simplicity, site specificity, high efficiency, and versatility. PMID:27170066

  15. Optimization Under Uncertainty of Site-Specific Turbine Configurations

    NASA Astrophysics Data System (ADS)

    Quick, J.; Dykes, K.; Graf, P.; Zahle, F.

    2016-09-01

    Uncertainty affects many aspects of wind energy plant performance and cost. In this study, we explore opportunities for site-specific turbine configuration optimization that accounts for uncertainty in the wind resource. As a demonstration, a simple empirical model for wind plant cost of energy is used in an optimization under uncertainty to examine how different risk appetites affect the optimal selection of a turbine configuration for sites of different wind resource profiles. If there is unusually high uncertainty in the site wind resource, the optimal turbine configuration diverges from the deterministic case and a generally more conservative design is obtained with increasing risk aversion on the part of the designer.

  16. Site specificity of adrenalectomy-induced brain growth.

    PubMed

    Thomas, T L; Devenport, L D

    1988-12-01

    Infant, juvenile, and adult brain growth is modulated by corticosterone. This study was designed to determine whether such modulation is confined to certain specific brain areas, and if the pattern of growth revealed is consistent across strains of rats. Young female Sprague-Dawley-derived rats were either adrenalectomized (ADX) or sham-operated (Sham) and allowed to mature 45 days before they were sacrificed for histological analysis. Fore brain sections were taken at several planes for display by projection microscope. Of the 21 sites examined, ADX exerted its greatest effect upon neocortical tissue and myelinated fiber tracts. The only other brain region affected was thalamus, which exhibited a significant widening as a result of ADX. In contrast, archicortical structures were notably unaffected by ADX. Neither the hippocampus, measured from a variety of planes, nor nuclei in the septal area were subject to increased growth by ADX. This general portrayal of ADX's site specificity held across strains of rats. However, there were local differences. Within the neopallium, the frontal region underwent the greatest thickening in one strain, while the occipital area was most strongly affected in the other. Parietal cortex was equally responsive in both strains. The pattern of sensitive vs insensitive sites bore a resemblance to the pattern of increased growth brought about by environmental enrichment as well as the fore brain distribution of Type 2 corticosterone receptors.

  17. Identification of Bacteria Synthesizing Ribosomal RNA in Response to Uranium Addition During Biostimulation at the Rifle, CO Integrated Field Research Site

    PubMed Central

    McGuinness, Lora R.; Wilkins, Michael J.; Williams, Kenneth H.; Long, Philip E.; Kerkhof, Lee J.

    2015-01-01

    Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this study, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate. The active microbes in the planktonic phase were deduced by monitoring ribosomes production via RT-PCR. The results indicated several microorganisms were synthesizing ribosomes in proportion with uranium amendment up to 2 μM. Concentrations of U (VI) >2 μM were generally found to inhibit ribosome synthesis. Two active bacteria responding to uranium addition in the field were close relatives of Desulfobacter postgateii and Geobacter bemidjiensis. Since RNA content often increases with growth rate, our findings suggest it is possible to rapidly elucidate active bacteria responding to the addition of uranium in field samples and provides a more targeted approach to stimulate specific populations to enhance radionuclide reduction in contaminated sites. PMID:26382047

  18. DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II)

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

    Bellon, S.F.; Coleman, J.H.; Lippard, S.J.

    The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedi-chloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-(Pt(NH{sub 3}){sub 2}(d(GpG))), cis-(Pt(NH){sub 3}{sub 2}(d(ApG))), and cis-(Pt(NH{sub 3}){sub 2}(d(GpTpG))) adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG. Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis. The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymersmore » containing the adducts. The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained. The authors find that the cis-GG and cis-AG adducts both unwind DNA by 13{degrees}, while the cis-GTG adduct unwinds DNA by 23{degrees}. In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts. On the basis of an analysis of the present and other published studies of site-specifically modified DNA. The authors propose that local duplex unwinding is a major determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease. In addition, local duplex unwinding of 13{degrees} and bending by 35{degrees} are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.« less

  19. 77 FR 65374 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-26

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory... management in the areas of environmental restoration, waste management, and related activities. Tentative...

  20. 76 FR 68179 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-03

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... November 14, 2011, of the Environmental Management Site-Specific Advisory Board, Idaho National Laboratory...: Robert L. Pence, Federal Coordinator, Department of Energy, Idaho Operations Office, 1955 Fremont Avenue...

  1. 19. Photographic copy of an asconstructed site plan for additions ...

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

    19. Photographic copy of an as-constructed site plan for additions to North Base: Job No. Muroc A(511), Military Construction, Third District Region, San Bernardino, California; Muroc Bombing Range, Muroc Lake, Calif; Additional Temporary Construction, Materiel Center Flight Test Base, Location Grading & Paving Plan, Sheet No. 1 of 21, March 1943. Reproduced from the holdings of the National Archives, Pacific Southwest Region - Edwards Air Force Base, North Base, North Base Road, Boron, Kern County, CA

  2. Optimization Under Uncertainty of Site-Specific Turbine Configurations

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

    Quick, J.; Dykes, K.; Graf, P.

    Uncertainty affects many aspects of wind energy plant performance and cost. In this study, we explore opportunities for site-specific turbine configuration optimization that accounts for uncertainty in the wind resource. As a demonstration, a simple empirical model for wind plant cost of energy is used in an optimization under uncertainty to examine how different risk appetites affect the optimal selection of a turbine configuration for sites of different wind resource profiles. Lastly, if there is unusually high uncertainty in the site wind resource, the optimal turbine configuration diverges from the deterministic case and a generally more conservative design is obtainedmore » with increasing risk aversion on the part of the designer.« less

  3. Optimization Under Uncertainty of Site-Specific Turbine Configurations

    DOE PAGES

    Quick, J.; Dykes, K.; Graf, P.; ...

    2016-10-03

    Uncertainty affects many aspects of wind energy plant performance and cost. In this study, we explore opportunities for site-specific turbine configuration optimization that accounts for uncertainty in the wind resource. As a demonstration, a simple empirical model for wind plant cost of energy is used in an optimization under uncertainty to examine how different risk appetites affect the optimal selection of a turbine configuration for sites of different wind resource profiles. Lastly, if there is unusually high uncertainty in the site wind resource, the optimal turbine configuration diverges from the deterministic case and a generally more conservative design is obtainedmore » with increasing risk aversion on the part of the designer.« less

  4. 77 FR 76475 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-28

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Northern New Mexico... the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal... INFORMATION CONTACT: Menice Santistevan, Northern New Mexico Citizens' Advisory Board (NNMCAB), 94 Cities of...

  5. 75 FR 24685 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-05

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory... prior to the meeting. ADDRESSES: Hilton Garden Inn, 700 Lindsay Boulevard, Idaho Falls, Idaho 83402. FOR...

  6. 77 FR 38276 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-27

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National... times prior to the meeting. ADDRESSES: Red Lion Hotel, 1555 Pocatello Creek Road, Pocatello, Idaho 83201...

  7. 76 FR 11773 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-03

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Northern New Mexico... the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal... Courtyard by Marriott, 3347 Cerrillos Road, Santa Fe, New Mexico 87507. FOR FURTHER INFORMATION CONTACT...

  8. LineageSpecificSeqgen: generating sequence data with lineage-specific variation in the proportion of variable sites

    PubMed Central

    Grievink, Liat Shavit; Penny, David; Hendy, Mike D; Holland, Barbara R

    2009-01-01

    Correction to Shavit Grievink L, Penny D, Hendy MD, Holland BR: LineageSpecificSeqgen: generating sequence data with lineage-specific variation in the proportion of variable sites. BMC Evol Biol 2008, 8(1):317.

  9. 75 FR 64719 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-20

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Northern New Mexico... the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal... at Santa Fe, 750 North St. Francis Drive, Santa Fe, New Mexico. FOR FURTHER INFORMATION CONTACT...

  10. 76 FR 22090 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-20

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Northern New Mexico... the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal... a.m.-5 p.m. ADDRESSES: Santa Claran Hotel, 464 North Riverside Drive, Espanola, New Mexico 87532...

  11. 75 FR 56527 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-16

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory... prior to the meeting. ADDRESSES: Coeur d'Alene Resort, 115 South Second Street, Coeur d'Alene, Idaho...

  12. 75 FR 53280 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-31

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Northern New Mexico... the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal... Inn and Conference Center, 1508 Paseo Del Pueblo Sur, Taos, New Mexico 87571. FOR FURTHER INFORMATION...

  13. Site specific modification of the human plasma proteome by methylglyoxal

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

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-argininemore » modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  14. Annotated Administrative Record Site-Specific Document Index, American Drum & Pallet Co. Removal Site, Memphis, Shelby County, Tennessee

    EPA Pesticide Factsheets

    Contains annotated index of site specific documents for the American Drum & Pallet Co. Removal Site in Memphis, Shelby County, Tennessee, January 9, 2008 Region ID: 04 DocID: 10517016, DocDate: 01-09-2008

  15. Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

    PubMed

    Cook, Brendon E; Adumeau, Pierre; Membreno, Rosemery; Carnazza, Kathryn E; Brand, Christian; Reiner, Thomas; Agnew, Brian J; Lewis, Jason S; Zeglis, Brian M

    2016-08-17

    In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.

  16. 75 FR 11872 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National... Site- Specific Advisory Board, Idaho National Laboratory to be held on March 16, 2010 75 FR 9590. In that notice, the meeting address was Hilton Garden Inn, 700 Lindsay Boulevard, Idaho Falls, Idaho 83402...

  17. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    PubMed

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  18. Specific phospholipid binding to Na,K-ATPase at two distinct sites.

    PubMed

    Habeck, Michael; Kapri-Pardes, Einat; Sharon, Michal; Karlish, Steven J D

    2017-03-14

    Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (α 1 β 1 ). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting E 1 P-E 2 P conformational transition (site B). We discuss the potential physiological implications.

  19. Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi.

    PubMed

    Hong, Yang; Hondalus, Mary K

    2008-10-01

    Streptomyces PhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

  20. 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

  1. Evaluating Protein Structure and Dynamics Using Co-Solvents, Photochemical Triggers, and Site-Specific Spectroscopic Probes

    NASA Astrophysics Data System (ADS)

    Abaskharon, Rachel M.

    As ubiquitous and diverse biopolymers, proteins are dynamic molecules that are constantly engaging in inter- and intramolecular interactions responsible for their structure, fold, and function. Because of this, gaining a comprehensive understanding of the factors that control protein conformation and dynamics remains elusive as current experimental techniques often lack the ability to initiate and probe a specific interaction or conformational transition. For this reason, this thesis aims to develop methods to control and monitor protein conformations, conformational transitions, and dynamics in a site-specific manner, as well as to understand how specific and non-specific interactions affect the protein folding energy landscape. First, by using the co-solvent, trifluoroethanol (TFE), we show that the rate at which a peptide folds can be greatly impacted and thus controlled by the excluded volume effect. Secondly, we demonstrate the utility of several light-responsive molecules and reactions as methods to manipulate and investigate protein-folding processes. Using an azobenzene linker as a photo-initiator, we are able to increase the folding rate of a protein system by an order of magnitude by channeling a sub-population through a parallel, faster folding pathway. Additionally, we utilize a tryptophan-mediated electron transfer process to a nearby disulfide bond to strategically unfold a protein molecule with ultraviolet light. We also demonstrate the potential of two ruthenium polypyridyl complexes as ultrafast phototriggers of protein reactions. Finally, we develop several site-specific spectroscopic probes of protein structure and environment. Specifically, we demonstrate that a 13C-labeled aspartic acid residue constitutes a useful site-specific infrared probe for investigating salt-bridges and hydration dynamics of proteins, particularly in proteins containing several acidic amino acids. We also show that a proline-derivative, 4-oxoproline, possesses novel

  2. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific.

    PubMed

    Thune, I; Furberg, A S

    2001-06-01

    The association between physical activity and overall and site-specific cancer risk is elaborated in relation to whether any observed dose-response association between physical activity and cancer can be interpreted in terms of how much physical activity (type, intensity, duration, frequency) is needed to influence site- and gender-specific cancer risk. Observational studies were reviewed that have examined the independent effect of the volume of occupational physical activity (OPA) and/or leisure time physical activity (LPA) on overall and site-specific cancer risk. The evidence of cohort and case-control studies suggests that both leisure time and occupational physical activity protect against overall cancer risk, with a graded dose-response association suggested in both sexes. Confounding effects such as diet, body weight, and parity are often included as a covariate in the analyses, with little influence on the observed associations. A crude graded inverse dose-response association was observed between physical activity and colon cancer in 48 studies including 40,674 colon/colorectal cancer cases for both sexes. A dose-response effect of physical activity on colon cancer risk was especially observed, when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed as MET-hours per week. An observed inverse association with a dose-response relationship between physical activity and breast cancer was also identified in the majority of the 41 studies including 108,031 breast cancer cases. The dose-response relationship was in particular observed in case-control studies and supported by observations in cohort studies when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed by MET-hours per week. This association between physical activity and breast cancer risk is possibly dependent on age at exposure, age at diagnosis, menopausal status and other effect

  3. Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies

    PubMed Central

    Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

    2015-01-01

    Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Conclusion: Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage. PMID:26605008

  4. Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies.

    PubMed

    Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

    2015-01-01

    Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage.

  5. Identification of bacteria synthesizing ribosomal RNA in response to uranium addition during biostimulation at the Rifle, CO Integrated Field Research site

    DOE PAGES

    McGuinness, Lora R.; Wilkins, Michael J.; Williams, Kenneth H.; ...

    2015-09-18

    Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this research, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate. The active microbes in the planktonic phase were deduced by monitoring ribosomes production via RT-PCR. The results indicated several microorganisms were synthesizing ribosomes in proportion with uranium amendment up to 2 μM. Concentrations of U (VI) >2 μM were generally found to inhibit ribosome synthesis. Two activemore » bacteria responding to uranium addition in the field were close relatives of Desulfobacter postgateii and Geobacter bemidjiensis. Since RNA content often increases with growth rate, our findings suggest it is possible to rapidly elucidate active bacteria responding to the addition of uranium in field samples and provides a more targeted approach to stimulate specific populations to enhance radionuclide reduction in contaminated sites.« less

  6. Site specific atomic polarizabilities in endohedral fullerenes and carbon onions

    NASA Astrophysics Data System (ADS)

    Zope, Rajendra R.; Bhusal, Shusil; Basurto, Luis; Baruah, Tunna; Jackson, Koblar

    2015-08-01

    We investigate the polarizability of trimetallic nitride endohedral fullerenes by partitioning the total polarizability into site specific components. This analysis indicates that the polarizability of the endohedral fullerene is essentially due to the outer fullerene cage and has insignificant contribution from the encapsulated unit. Thus, the outer fullerene cages effectively shield the encapsulated clusters and behave like Faraday cages. The polarizability of endohedral fullerenes is slightly smaller than the polarizability of the corresponding bare carbon fullerenes. The application of the site specific polarizabilities to C60@C240 and C60@C180 onions shows that, compared to the polarizability of isolated C60 fullerene, the encapsulation of the C60 in C240 and C180 fullerenes reduces its polarizability by 75% and 83%, respectively. The differences in the polarizability of C60 in the two onions is a result of differences in the bonding (intershell electron transfer), fullerene shell relaxations, and intershell separations. The site specific analysis further shows that the outer atoms in a fullerene shell contribute most to the fullerene polarizability.

  7. Site specific atomic polarizabilities in endohedral fullerenes and carbon onions.

    PubMed

    Zope, Rajendra R; Bhusal, Shusil; Basurto, Luis; Baruah, Tunna; Jackson, Koblar

    2015-08-28

    We investigate the polarizability of trimetallic nitride endohedral fullerenes by partitioning the total polarizability into site specific components. This analysis indicates that the polarizability of the endohedral fullerene is essentially due to the outer fullerene cage and has insignificant contribution from the encapsulated unit. Thus, the outer fullerene cages effectively shield the encapsulated clusters and behave like Faraday cages. The polarizability of endohedral fullerenes is slightly smaller than the polarizability of the corresponding bare carbon fullerenes. The application of the site specific polarizabilities to C60@C240 and C60@C180 onions shows that, compared to the polarizability of isolated C60 fullerene, the encapsulation of the C60 in C240 and C180 fullerenes reduces its polarizability by 75% and 83%, respectively. The differences in the polarizability of C60 in the two onions is a result of differences in the bonding (intershell electron transfer), fullerene shell relaxations, and intershell separations. The site specific analysis further shows that the outer atoms in a fullerene shell contribute most to the fullerene polarizability.

  8. Site-Specific Biomolecule Labeling with Gold Clusters

    PubMed Central

    Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

    2013-01-01

    Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (His)-tagged proteins. PMID:20887859

  9. SPEER-SERVER: a web server for prediction of protein specificity determining sites

    PubMed Central

    Chakraborty, Abhijit; Mandloi, Sapan; Lanczycki, Christopher J.; Panchenko, Anna R.; Chakrabarti, Saikat

    2012-01-01

    Sites that show specific conservation patterns within subsets of proteins in a protein family are likely to be involved in the development of functional specificity. These sites, generally termed specificity determining sites (SDS), might play a crucial role in binding to a specific substrate or proteins. Identification of SDS through experimental techniques is a slow, difficult and tedious job. Hence, it is very important to develop efficient computational methods that can more expediently identify SDS. Herein, we present Specificity prediction using amino acids’ Properties, Entropy and Evolution Rate (SPEER)-SERVER, a web server that predicts SDS by analyzing quantitative measures of the conservation patterns of protein sites based on their physico-chemical properties and the heterogeneity of evolutionary changes between and within the protein subfamilies. This web server provides an improved representation of results, adds useful input and output options and integrates a wide range of analysis and data visualization tools when compared with the original standalone version of the SPEER algorithm. Extensive benchmarking finds that SPEER-SERVER exhibits sensitivity and precision performance that, on average, meets or exceeds that of other currently available methods. SPEER-SERVER is available at http://www.hpppi.iicb.res.in/ss/. PMID:22689646

  10. SPEER-SERVER: a web server for prediction of protein specificity determining sites.

    PubMed

    Chakraborty, Abhijit; Mandloi, Sapan; Lanczycki, Christopher J; Panchenko, Anna R; Chakrabarti, Saikat

    2012-07-01

    Sites that show specific conservation patterns within subsets of proteins in a protein family are likely to be involved in the development of functional specificity. These sites, generally termed specificity determining sites (SDS), might play a crucial role in binding to a specific substrate or proteins. Identification of SDS through experimental techniques is a slow, difficult and tedious job. Hence, it is very important to develop efficient computational methods that can more expediently identify SDS. Herein, we present Specificity prediction using amino acids' Properties, Entropy and Evolution Rate (SPEER)-SERVER, a web server that predicts SDS by analyzing quantitative measures of the conservation patterns of protein sites based on their physico-chemical properties and the heterogeneity of evolutionary changes between and within the protein subfamilies. This web server provides an improved representation of results, adds useful input and output options and integrates a wide range of analysis and data visualization tools when compared with the original standalone version of the SPEER algorithm. Extensive benchmarking finds that SPEER-SERVER exhibits sensitivity and precision performance that, on average, meets or exceeds that of other currently available methods. SPEER-SERVER is available at http://www.hpppi.iicb.res.in/ss/.

  11. Human disturbance and stage-specific habitat requirements influence snowy plover site occupancy during the breeding season

    PubMed Central

    Webber, Alyson F; Heath, Julie A; Fischer, Richard A

    2013-01-01

    Habitat use has important consequences for avian reproductive success and survival. In coastal areas with recreational activity, human disturbance may limit use of otherwise suitable habitat. Snowy plovers Charadrius nivosus have a patchy breeding distribution along the coastal areas on the Florida Panhandle, USA. Our goal was to determine the relative effects of seasonal human disturbance and habitat requirements on snowy plover habitat use. We surveyed 303 sites for snowy plovers, human disturbance, and habitat features between January and July 2009 and 2010. We made multiple visits during three different sampling periods that corresponded to snowy plover breeding: pre-breeding, incubation, and brood-rearing and used multi-season occupancy models to examine whether human disturbance, habitat features, or both influenced site occupancy, colonization (probability of transition from an unoccupied site to an occupied site), and extinction (probability of transition from an occupied site to an unoccupied site). Snowy plover site occupancy and colonization was negatively associated with human disturbance and site extinction was positively associated with human disturbance. Interdune vegetation had a negative effect on occupancy and colonization, indicating that plovers were less likely to use areas with uniform, dense vegetation among dunes. Also, dune shape, beach debris, and access to low-energy foraging areas influenced site occupancy, colonization, and extinction. Plovers used habitat based on beach characteristics that provided stage-specific resource needs; however, human disturbance was the strongest predictor of site occupancy. In addition, vegetation plantings used to enhance dune rehabilitation may negatively impact plover site occupancy. Management actions that decrease human disturbance, such as symbolic fencing and signage, may increase the amount of breeding habitat available to snowy plovers on the Florida Panhandle and in other areas with high human

  12. Adapter reagents for protein site specific dye labeling.

    PubMed

    Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E

    2014-05-01

    Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc.

  13. Adapter Reagents for Protein Site Specific Dye Labeling

    PubMed Central

    Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

    2016-01-01

    Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

  14. Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

    PubMed Central

    Antos, John M.; Ingram, Jessica; Fang, Tao; Pishesha, Novalia; Truttmann, Matthias C.; Ploegh, Hidde L.

    2017-01-01

    Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five amino acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. PMID:19365788

  15. Active Site Mutations Change the Cleavage Specificity of Neprilysin

    PubMed Central

    Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

    2012-01-01

    Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

  16. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    PubMed

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  17. A systematic identification of species-specific protein succinylation sites using joint element features information.

    PubMed

    Hasan, Md Mehedi; Khatun, Mst Shamima; Mollah, Md Nurul Haque; Yong, Cao; Guo, Dianjing

    2017-01-01

    Lysine succinylation, an important type of protein posttranslational modification, plays significant roles in many cellular processes. Accurate identification of succinylation sites can facilitate our understanding about the molecular mechanism and potential roles of lysine succinylation. However, even in well-studied systems, a majority of the succinylation sites remain undetected because the traditional experimental approaches to succinylation site identification are often costly, time-consuming, and laborious. In silico approach, on the other hand, is potentially an alternative strategy to predict succinylation substrates. In this paper, a novel computational predictor SuccinSite2.0 was developed for predicting generic and species-specific protein succinylation sites. This predictor takes the composition of profile-based amino acid and orthogonal binary features, which were used to train a random forest classifier. We demonstrated that the proposed SuccinSite2.0 predictor outperformed other currently existing implementations on a complementarily independent dataset. Furthermore, the important features that make visible contributions to species-specific and cross-species-specific prediction of protein succinylation site were analyzed. The proposed predictor is anticipated to be a useful computational resource for lysine succinylation site prediction. The integrated species-specific online tool of SuccinSite2.0 is publicly accessible.

  18. [Case study on health risk assessment based on site-specific conceptual model].

    PubMed

    Zhong, Mao-Sheng; Jiang, Lin; Yao, Jue-Jun; Xia, Tian-Xiang; Zhu, Xiao-Ying; Han, Dan; Zhang, Li-Na

    2013-02-01

    Site investigation was carried out on an area to be redeveloped as a subway station, which is right downstream of the groundwater of a former chemical plant. The results indicate the subsurface soil and groundwater in the area are both polluted heavily by 1,2-dichloroethane, which was caused by the chemical plant upstream with the highest concentration was 104.08 mg.kg-1 for soil sample at 8.6 m below ground and the highest concentration was 18500 microg.L-1 for groundwater. Further, a site-specific contamination conceptual model, giving consideration to the specific structure configuration of the station, was developed, and the corresponding risk calculation equation was derived. The carcinogenic risks calculated with models developed on the generic site conceptual model and derived herein on the site-specific conceptual model were compared. Both models indicate that the carcinogenic risk is significantly higher than the acceptable level which is 1 x 10(-6). The comparison result reveals that the risk calculated with the former models for soil and groundwater are higher than the one calculated with the latter models by 2 times and 1.5 times, respectively. The finding in this paper indicates that the generic risk assessment model may underestimate the risk if specific site conditions and structure configuration are not considered.

  19. Report: EPA Could Improve Its Redistribution of Superfund Payments to Specific Sites

    EPA Pesticide Factsheets

    Report #2006-P-00027, July 31, 2006. EPA did not make timely redistributions of Superfund coop agreement, interagency agreement, and small purchase payments from the general site identifier “WQ” to the specific Superfund sites or other site identifiers.

  20. CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.

    PubMed

    Fedoreyeva, L I; Vanyushin, B F

    2011-06-01

    Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.

  1. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    PubMed

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  2. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  3. 76 FR 30027 - Land Disposal Restrictions: Site-Specific Treatment Variance for Hazardous Selenium-Bearing Waste...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-24

    ... Restrictions: Site-Specific Treatment Variance for Hazardous Selenium-Bearing Waste Treated by U.S. Ecology.... Ecology Nevada in Beatty, Nevada and withdrew an existing site- specific treatment variance issued to... 268.44(o)) by granting a site-specific treatment variance to U.S. Ecology Nevada in Beatty, Nevada and...

  4. Site specific atomic polarizabilities in endohedral fullerenes and carbon onions

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

    Zope, Rajendra R., E-mail: rzope@utep.edu; Baruah, Tunna; Computational Science Program, The University of Texas at El Paso, El Paso, Texas 79958

    2015-08-28

    We investigate the polarizability of trimetallic nitride endohedral fullerenes by partitioning the total polarizability into site specific components. This analysis indicates that the polarizability of the endohedral fullerene is essentially due to the outer fullerene cage and has insignificant contribution from the encapsulated unit. Thus, the outer fullerene cages effectively shield the encapsulated clusters and behave like Faraday cages. The polarizability of endohedral fullerenes is slightly smaller than the polarizability of the corresponding bare carbon fullerenes. The application of the site specific polarizabilities to C{sub 60}@C{sub 240} and C{sub 60}@C{sub 180} onions shows that, compared to the polarizability of isolatedmore » C{sub 60} fullerene, the encapsulation of the C{sub 60} in C{sub 240} and C{sub 180} fullerenes reduces its polarizability by 75% and 83%, respectively. The differences in the polarizability of C{sub 60} in the two onions is a result of differences in the bonding (intershell electron transfer), fullerene shell relaxations, and intershell separations. The site specific analysis further shows that the outer atoms in a fullerene shell contribute most to the fullerene polarizability.« less

  5. Site-specific biomolecule labeling with gold clusters.

    PubMed

    Ackerson, Christopher J; Powell, Richard D; Hainfeld, James F

    2010-01-01

    Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: linker-mediated bioconjugation, direct gold-biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (His)-tagged proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.

    PubMed

    Moriuchi, Hiromi; Unno, Hideaki; Goda, Shuichiro; Tateno, Hiroaki; Hirabayashi, Jun; Hatakeyama, Tomomitsu

    2015-07-01

    CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Site-specific protein labeling with PRIME and chelation-assisted Click chemistry

    PubMed Central

    Uttamapinant, Chayasith; Sanchez, Mateo I.; Liu, Daniel S.; Yao, Jennifer Z.; White, Katharine A.; Grecian, Scott; Clarke, Scott; Gee, Kyle R.; Ting, Alice Y.

    2016-01-01

    This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: PRobe Incorporation Mediated by Enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase site-specifically attaches a picolyl azide derivative to a 13-amino acid recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing picolyl azide are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize picolyl azide, perform PRIME labeling, and achieve CuAAC derivatization of picolyl azide on live cells, fixed cells, and purified proteins. Reagent preparations, including synthesis of picolyl azide probes and expression of lipoic acid ligase, take 12 d, while the procedure to perform site-specific picolyl azide ligation and CuAAC on cells or on purified proteins takes 40 min-3 h. PMID:23887180

  8. Optimization under Uncertainty of Site-Specific Turbine Configurations: Preprint

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

    Quick, Julian; Dykes, Katherine; Graf, Peter

    Uncertainty affects many aspects of wind energy plant performance and cost. In this study, we explore opportunities for site-specific turbine configuration optimization that accounts for uncertainty in the wind resource. As a demonstration, a simple empirical model for wind plant cost of energy is used in an optimization under uncertainty to examine how different risk appetites affect the optimal selection of a turbine configuration for sites of different wind resource profiles. If there is unusually high uncertainty in the site wind resource, the optimal turbine configuration diverges from the deterministic case and a generally more conservative design is obtained withmore » increasing risk aversion on the part of the designer.« less

  9. Hetero-site-specific X-ray pump-probe spectroscopy for femtosecond intramolecular dynamics

    DOE PAGES

    Picón, A.; Lehmann, C. S.; Bostedt, C.; ...

    2016-05-23

    New capabilities at X-ray free-electron laser facilities allow the generation of two-colour femtosecond X-ray pulses, opening the possibility of performing ultrafast studies of X-ray-induced phenomena. Specifically, the experimental realization of hetero-site-specific X-ray-pump/X-ray-probe spectroscopy is of special interest, in which an X-ray pump pulse is absorbed at one site within a molecule and an X-ray probe pulse follows the X-ray-induced dynamics at another site within the same molecule. In this paper, we show experimental evidence of a hetero-site pump-probe signal. By using two-colour 10-fs X-ray pulses, we are able to observe the femtosecond time dependence for the formation of F ionsmore » during the fragmentation of XeF 2 molecules following X-ray absorption at the Xe site.« less

  10. Generalized theory on the mechanism of site-specific DNA-protein interactions

    NASA Astrophysics Data System (ADS)

    Niranjani, G.; Murugan, R.

    2016-05-01

    We develop a generalized theoretical framework on the binding of transcription factor proteins (TFs) with specific sites on DNA that takes into account the interplay of various factors regarding overall electrostatic potential at the DNA-protein interface, occurrence of kinetic traps along the DNA sequence, presence of other roadblock protein molecules along DNA and crowded environment, conformational fluctuations in the DNA binding domains (DBDs) of TFs, and the conformational state of the DNA. Starting from a Smolochowski type theoretical framework on site-specific binding of TFs we logically build our model by adding the effects of these factors one by one. Our generalized two-step model suggests that the electrostatic attractive forces present inbetween the positively charged DBDs of TFs and the negatively charged phosphate backbone of DNA, along with the counteracting shielding effects of solvent ions, is the core factor that creates a fluidic type environment at the DNA-protein interface. This in turn facilitates various one-dimensional diffusion (1Dd) processes such as sliding, hopping and intersegmental transfers. These facilitating processes as well as flipping dynamics of conformational states of DBDs of TFs between stationary and mobile states can enhance the 1Dd coefficient on a par with three-dimensional diffusion (3Dd). The random coil conformation of DNA also plays critical roles in enhancing the site-specific association rate. The extent of enhancement over the 3Dd controlled rate seems to be directly proportional to the maximum possible 1Dd length. We show that the overall site-specific binding rate scales with the length of DNA in an asymptotic way. For relaxed DNA, the specific binding rate will be independent of the length of DNA as length increases towards infinity. For condensed DNA as in in vivo conditions, the specific binding rate depends on the length of DNA in a turnover way with a maximum. This maximum rate seems to scale with the

  11. Site-specific equilibrium isotopic fractionation of oxygen, carbon and calcium in apatite

    NASA Astrophysics Data System (ADS)

    Aufort, Julie; Ségalen, Loïc; Gervais, Christel; Paulatto, Lorenzo; Blanchard, Marc; Balan, Etienne

    2017-12-01

    The stable isotope composition of biogenic apatite is an important geochemical marker that can record environmental parameters and is widely used to infer past climates, biomineralization processes, dietary preferences and habitat of vertebrates. In this study, theoretical equilibrium isotopic fractionation of oxygen, carbon and calcium in hydroxyapatite and carbonate-bearing hydroxyapatite is investigated using first-principles methods based on density-functional theory and compared to the theoretical isotopic fractionation properties of calcite, CO2 and H2O. Considering the variability of apatite crystal-chemistry, special attention is given to specific contributions of crystal sites to isotopic fractionation. Significant internal fractionation is calculated for oxygen and carbon isotopes in CO3 between the different structural sites occupied by carbonate groups in apatite (typically 7‰ for both 18O/16O and 13C/12C fractionation at 37 °C). Compared with calcite-water oxygen isotope fractionation, occurrence of A-type substitution in apatite structure, in addition to the main B-type substitution, could explain the larger temperature dependence of oxygen isotope fractionation measured at low temperature between carbonate in apatite and water. Theoretical internal fractionation of oxygen isotopes between carbonate and phosphate in B-type carbonated apatite (∼8‰ at 37 °C) is consistent with experimental values obtained from modern and well-preserved fossil bio-apatites. Concerning calcium, theoretical results suggest a small fractionation between apatite and calcite (-0.17‰ at 37 °C). Internal fractionation reaching 0.8‰ at 37 °C occurs between the two Ca sites in hydroxyapatite. Furthermore, the Ca isotopic fractionation properties of apatite are affected by the occurrence of carbonate groups, which could contribute to the variability observed on natural samples. Owing to the complexity of apatite crystal-chemistry and in light of the theoretical

  12. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    PubMed

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. Copyright © 2016. Published by Elsevier B.V.

  13. The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

    PubMed Central

    Jasin, Maria; Haber, James E.

    2017-01-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. PMID:27261202

  14. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins*

    PubMed Central

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-01-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... management plan, and addition of multiple-site components. 921.33 Section 921.33 Commerce and Foreign Trade... management plan, and addition of multiple-site components. (a) Changes in the boundary of a Reserve and major... management plan shall address goals and objectives for all components of the multi-site Reserve and the...

  16. Single-Stranded γPNAs for In Vivo Site-Specific Genome Editing via Watson-Crick Recognition

    PubMed Central

    Bahal, Raman; Quijano, Elias; McNeer, Nicole Ali; Liu, Yanfeng; Bhunia, Dinesh C.; López-Giráldez, Francesco; Fields, Rachel J.; Saltzman, W. Mark; Ly, Danith H.; Glazer, Peter M.

    2014-01-01

    Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction. PMID:25174576

  17. Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

    PubMed

    Bahal, Raman; Quijano, Elias; McNeer, Nicole A; Liu, Yanfeng; Bhunia, Dinesh C; Lopez-Giraldez, Francesco; Fields, Rachel J; Saltzman, William M; Ly, Danith H; Glazer, Peter M

    2014-01-01

    Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

  18. Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure

    PubMed Central

    Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

    2014-01-01

    Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a “site-specific” homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional “non-site-specific” allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view. PMID:24710521

  19. Site-specific thromboembolism: a novel animal model for stroke.

    PubMed

    Ringer, Andrew J; Guterman, Lee R; Hopkins, L Nelson

    2004-02-01

    To develop a technique for site-specific placement of a thrombus of predetermined volume in an animal model for the purpose of evaluating methods of intravascular thrombolysis and clot retrieval. Six swine were subjected to thrombus injection bilaterally in the ascending pharyngeal artery (APA). Each animal underwent transfemoral angiography while under general anesthesia. A nondetachable balloon catheter and a 3-French microcatheter were then advanced into the common carotid artery through a 7-French guide catheter. With the microcatheter in the proximal APA and the balloon inflated proximally, a bolus of preformed thrombus composed of 0.9 mL of autologous blood and 0.1 mL of bovine thrombin (200 IU/mL) was injected through the microcatheter while local flow arrest was maintained for 15 min. The balloon was deflated and removed. The occluded arteries were observed by serial angiography for 3 hr and then resected for gross examination and hematoxylin and eosin staining. Each APA was occluded angiographically and did not recanalize during the 3-hr observation period. Persistent, proximal progression of thrombus to the superior thyroid artery origin occurred in three animals. Gross inspection revealed that the resected arteries contained thrombus in the proximal APA but not in the common carotid artery. Histologic examination revealed organized thrombus, without evidence of intimal injury. Our model provides a simple, reliable method for site-specific injection of a thrombus of predetermined volume. Site-specific placement is important for evaluation of the efficacy of thrombolytic agents and techniques. Angiographic evidence of brain revascularization can be used to grade revascularization and clot volume. The ability to specifically localize and estimate clot volume makes our model well suited for the evaluation and comparison of thrombolytic agents and endovascular techniques.

  20. Site Specific Probable Maximum Precipitation Estimates and Professional Judgement

    NASA Astrophysics Data System (ADS)

    Hayes, B. D.; Kao, S. C.; Kanney, J. F.; Quinlan, K. R.; DeNeale, S. T.

    2015-12-01

    State and federal regulatory authorities currently rely upon the US National Weather Service Hydrometeorological Reports (HMRs) to determine probable maximum precipitation (PMP) estimates (i.e., rainfall depths and durations) for estimating flooding hazards for relatively broad regions in the US. PMP estimates for the contributing watersheds upstream of vulnerable facilities are used to estimate riverine flooding hazards while site-specific estimates for small water sheds are appropriate for individual facilities such as nuclear power plants. The HMRs are often criticized due to their limitations on basin size, questionable applicability in regions affected by orographic effects, their lack of consist methods, and generally by their age. HMR-51 for generalized PMP estimates for the United States east of the 105th meridian, was published in 1978 and is sometimes perceived as overly conservative. The US Nuclear Regulatory Commission (NRC), is currently reviewing several flood hazard evaluation reports that rely on site specific PMP estimates that have been commercially developed. As such, NRC has recently investigated key areas of expert judgement via a generic audit and one in-depth site specific review as they relate to identifying and quantifying actual and potential storm moisture sources, determining storm transposition limits, and adjusting available moisture during storm transposition. Though much of the approach reviewed was considered a logical extension of HMRs, two key points of expert judgement stood out for further in-depth review. The first relates primarily to small storms and the use of a heuristic for storm representative dew point adjustment developed for the Electric Power Research Institute by North American Weather Consultants in 1993 in order to harmonize historic storms for which only 12 hour dew point data was available with more recent storms in a single database. The second issue relates to the use of climatological averages for spatially

  1. Evaluation of potential water conservation using site-specific irrigation

    USDA-ARS?s Scientific Manuscript database

    With the advent of site-specific variable-rate irrigation (VRI) systems, irrigation can be spatially managed within sub-field-sized zones. Spatial irrigation management can optimize spatial water use efficiency and may conserve water. Spatial VRI systems are currently being managed by consultants ...

  2. High-Affinity Quasi-Specific Sites in the Genome: How the DNA-Binding Proteins Cope with Them

    PubMed Central

    Chakrabarti, J.; Chandra, Navin; Raha, Paromita; Roy, Siddhartha

    2011-01-01

    Many prokaryotic transcription factors home in on one or a few target sites in the presence of a huge number of nonspecific sites. Our analysis of λ-repressor in the Escherichia coli genome based on single basepair substitution experiments shows the presence of hundreds of sites having binding energy within 3 Kcal/mole of the OR1 binding energy, and thousands of sites with binding energy above the nonspecific binding energy. The effect of such sites on DNA-based processes has not been fully explored. The presence of such sites dramatically lowers the occupation probability of the specific site far more than if the genome were composed of nonspecific sites only. Our Brownian dynamics studies show that the presence of quasi-specific sites results in very significant kinetic effects as well. In contrast to λ-repressor, the E. coli genome has orders of magnitude lower quasi-specific sites for GalR, an integral transcription factor, thus causing little competition for the specific site. We propose that GalR and perhaps repressors of the same family have evolved binding modes that lead to much smaller numbers of quasi-specific sites to remove the untoward effects of genomic DNA. PMID:21889449

  3. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    PubMed

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  4. CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo¶

    PubMed Central

    Deterding, Leesa J.; Williams, Jason G.; Humble, Margaret M.; Petrovich, Robert M.; Wei, Sung-Jen; Trempus, Carol S.; Gates, Matthew B.; Zhu, Feng; Smart, Robert C.; Tennant, Raymond W.; Tomer, Kenneth B.

    2010-01-01

    CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309–382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34. PMID:21499536

  5. Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation

    PubMed Central

    Kovacs, Izabella; Lindermayr, Christian

    2013-01-01

    Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress, and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation.In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein–protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms, and on structural analysis of cysteine S-nitrosylation. PMID:23717319

  6. Towards soft robotic devices for site-specific drug delivery.

    PubMed

    Alici, Gursel

    2015-01-01

    Considerable research efforts have recently been dedicated to the establishment of various drug delivery systems (DDS) that are mechanical/physical, chemical and biological/molecular DDS. In this paper, we report on the recent advances in site-specific drug delivery (site-specific, controlled, targeted or smart drug delivery are terms used interchangeably in the literature, to mean to transport a drug or a therapeutic agent to a desired location within the body and release it as desired with negligibly small toxicity and side effect compared to classical drug administration means such as peroral, parenteral, transmucosal, topical and inhalation) based on mechanical/physical systems consisting of implantable and robotic drug delivery systems. While we specifically focus on the robotic or autonomous DDS, which can be reprogrammable and provide multiple doses of a drug at a required time and rate, we briefly cover the implanted DDS, which are well-developed relative to the robotic DDS, to highlight the design and performance requirements, and investigate issues associated with the robotic DDS. Critical research issues associated with both DDSs are presented to describe the research challenges ahead of us in order to establish soft robotic devices for clinical and biomedical applications.

  7. 30 CFR 46.11 - Site-specific hazard awareness training.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Section 46.11 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND... workers; (4) Customers, including commercial over-the-road truck drivers; (5) Construction workers or... procedures. The training must address site-specific health and safety risks, such as unique geologic or...

  8. Adoption of site-specific variable rate sprinkler irrigation systems

    USDA-ARS?s Scientific Manuscript database

    More than twenty years of private and public research on site-specific variable-rate sprinkler irrigation (SS-VRI) technology has resulted in limited commercial adoption of the technology. Competing patents, liability and proprietary software have affected industry’s willingness to move into a new t...

  9. Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans.

    PubMed

    Noborn, Fredrik; Gomez Toledo, Alejandro; Green, Anders; Nasir, Waqas; Sihlbom, Carina; Nilsson, Jonas; Larson, Göran

    2016-10-03

    Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.

  10. Appreciating "Thirdspace": An Alternative Way of Viewing and Valuing Site-Specific Dance Performance

    ERIC Educational Resources Information Center

    Munjee, Tara

    2014-01-01

    Site-specific dance performance involves the presentation of choreography in connection with a site. The context of the site combined with a viewer's personal history, beliefs, and identity impact the reading and appreciation of the performance. Although both stage and site dance performance valuing elicit multiple interpretations of artistic…

  11. Probability-based estimates of site-specific copper water quality criteria for the Chesapeake Bay, USA.

    PubMed

    Arnold, W Ray; Warren-Hicks, William J

    2007-01-01

    The object of this study was to estimate site- and region-specific dissolved copper criteria for a large embayment, the Chesapeake Bay, USA. The intent is to show the utility of 2 copper saltwater quality site-specific criteria estimation models and associated region-specific criteria selection methods. The criteria estimation models and selection methods are simple, efficient, and cost-effective tools for resource managers. The methods are proposed as potential substitutes for the US Environmental Protection Agency's water effect ratio methods. Dissolved organic carbon data and the copper criteria models were used to produce probability-based estimates of site-specific copper saltwater quality criteria. Site- and date-specific criteria estimations were made for 88 sites (n = 5,296) in the Chesapeake Bay. The average and range of estimated site-specific chronic dissolved copper criteria for the Chesapeake Bay were 7.5 and 5.3 to 16.9 microg Cu/L. The average and range of estimated site-specific acute dissolved copper criteria for the Chesapeake Bay were 11.7 and 8.3 to 26.4 microg Cu/L. The results suggest that applicable national and state copper criteria can increase in much of the Chesapeake Bay and remain protective. Virginia Department of Environmental Quality copper criteria near the mouth of the Chesapeake Bay, however, need to decrease to protect species of equal or greater sensitivity to that of the marine mussel, Mytilus sp.

  12. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

    PubMed

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2018-01-01

    Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

  13. DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

    PubMed

    Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

    2013-01-01

    Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

  14. Detection of site specific glycosylation in proteins using flow cytometry†

    PubMed Central

    Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

    2009-01-01

    We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

  15. 36 CFR 6.6 - Solid waste disposal sites within new additions to the National Park System.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... within new additions to the National Park System. 6.6 Section 6.6 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SOLID WASTE DISPOSAL SITES IN UNITS OF THE NATIONAL PARK SYSTEM § 6.6 Solid waste disposal sites within new additions to the National Park System. (a) An operator...

  16. 36 CFR 6.6 - Solid waste disposal sites within new additions to the National Park System.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... within new additions to the National Park System. 6.6 Section 6.6 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SOLID WASTE DISPOSAL SITES IN UNITS OF THE NATIONAL PARK SYSTEM § 6.6 Solid waste disposal sites within new additions to the National Park System. (a) An operator...

  17. 36 CFR 6.6 - Solid waste disposal sites within new additions to the National Park System.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... within new additions to the National Park System. 6.6 Section 6.6 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SOLID WASTE DISPOSAL SITES IN UNITS OF THE NATIONAL PARK SYSTEM § 6.6 Solid waste disposal sites within new additions to the National Park System. (a) An operator...

  18. Unravelling site-specific breast cancer metastasis: a microRNA expression profiling study

    PubMed Central

    Schrijver, Willemijne A.M.E.; van Diest, Paul J.; Moelans, Cathy B

    2017-01-01

    Distant metastasis is still the main cause of death from breast cancer. MicroRNAs (miRs) are important regulators of many physiological and pathological processes, including metastasis. Molecular breast cancer subtypes are known to show a site-specific pattern of metastases formation. In this study, we set out to determine the underlying molecular mechanisms of site-specific breast cancer metastasis by microRNA expression profiling. To identify a miR signature for metastatic breast carcinoma that could predict metastatic localization, we compared global miR expression in 23 primary breast cancer specimens with their corresponding multiple distant metastases to ovary (n=9), skin (n=12), lung (n=10), brain (n=4) and gastrointestinal tract (n=10) by miRCURY microRNA expression arrays. For validation, we performed quantitative real-time (qRT) PCR on the discovery cohort and on an independent validation cohort of 29 primary breast cancer specimens and their matched metastases. miR expression was highly patient specific and miR signatures in the primary tumor were largely retained in the metastases, with the exception of several differentially expressed, location specific miRs. Validation with qPCR demonstrated that hsa-miR-106b-5p was predictive for the development of lung metastases. In time, the second metastasis often showed a miR upregulation compared to the first metastasis. This study discovered a metastatic site-specific miR and found miR expression to be highly patient specific. This may lead to novel biomarkers predicting site of distant metastases, and to adjuvant, personalized targeted therapy strategies that could prevent such metastases from becoming clinically manifest. PMID:27902972

  19. Unravelling site-specific breast cancer metastasis: a microRNA expression profiling study.

    PubMed

    Schrijver, Willemijne A M E; van Diest, Paul J; Moelans, Cathy B

    2017-01-10

    Distant metastasis is still the main cause of death from breast cancer. MicroRNAs (miRs) are important regulators of many physiological and pathological processes, including metastasis. Molecular breast cancer subtypes are known to show a site-specific pattern of metastases formation. In this study, we set out to determine the underlying molecular mechanisms of site-specific breast cancer metastasis by microRNA expression profiling.To identify a miR signature for metastatic breast carcinoma that could predict metastatic localization, we compared global miR expression in 23 primary breast cancer specimens with their corresponding multiple distant metastases to ovary (n=9), skin (n=12), lung (n=10), brain (n=4) and gastrointestinal tract (n=10) by miRCURY microRNA expression arrays. For validation, we performed quantitative real-time (qRT) PCR on the discovery cohort and on an independent validation cohort of 29 primary breast cancer specimens and their matched metastases.miR expression was highly patient specific and miR signatures in the primary tumor were largely retained in the metastases, with the exception of several differentially expressed, location specific miRs. Validation with qPCR demonstrated that hsa-miR-106b-5p was predictive for the development of lung metastases. In time, the second metastasis often showed a miR upregulation compared to the first metastasis.This study discovered a metastatic site-specific miR and found miR expression to be highly patient specific. This may lead to novel biomarkers predicting site of distant metastases, and to adjuvant, personalized targeted therapy strategies that could prevent such metastases from becoming clinically manifest.

  20. Identification of S-glutathionylation sites in species-specific proteins by incorporating five sequence-derived features into the general pseudo-amino acid composition.

    PubMed

    Zhao, Xiaowei; Ning, Qiao; Ai, Meiyue; Chai, Haiting; Yang, Guifu

    2016-06-07

    As a selective and reversible protein post-translational modification, S-glutathionylation generates mixed disulfides between glutathione (GSH) and cysteine residues, and plays an important role in regulating protein activity, stability, and redox regulation. To fully understand S-glutathionylation mechanisms, identification of substrates and specific S-Glutathionylated sites is crucial. Experimental identification of S-glutathionylated sites is labor-intensive and time consuming, so establishing an effective computational method is much desirable due to their convenient and fast speed. Therefore, in this study, a new bioinformatics tool named SSGlu (Species-Specific identification of Protein S-glutathionylation Sites) was developed to identify species-specific protein S-glutathionylated sites, utilizing support vector machines that combine multiple sequence-derived features with a two-step feature selection. By 5-fold cross validation, the performance of SSGlu was measured with an AUC of 0.8105 and 0.8041 for Homo sapiens and Mus musculus, respectively. Additionally, SSGlu was compared with the existing methods, and the higher MCC and AUC of SSGlu demonstrated that SSGlu was very promising to predict S-glutathionylated sites. Furthermore, a site-specific analysis showed that S-glutathionylation intimately correlated with the features derived from its surrounding sites. The conclusions derived from this study might help to understand more of the S-glutathionylation mechanism and guide the related experimental validation. For public access, SSGlu is freely accessible at http://59.73.198.144:8080/SSGlu/. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Virus-Specific Immune Memory at Peripheral Sites of Herpes Simplex Virus Type 2 (HSV-2) Infection in Guinea Pigs

    PubMed Central

    Xia, Jingya; Veselenak, Ronald L.; Gorder, Summer R.; Bourne, Nigel; Milligan, Gregg N.

    2014-01-01

    Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4+ and CD8+ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency

  2. 76 FR 24831 - Site-Specific Analyses for Demonstrating Compliance With Subpart C Performance Objectives

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-03

    ... available under ADAMS accession number ML111040419, and the ``Technical Analysis Supporting Definition of... NUCLEAR REGULATORY COMMISSION 10 CFR Part 61 RIN 3150-AI92 [NRC-2011-0012] Site-Specific Analyses...-level radioactive waste disposal facilities to conduct site-specific analyses to demonstrate compliance...

  3. PPSP: prediction of PK-specific phosphorylation site with Bayesian decision theory.

    PubMed

    Xue, Yu; Li, Ao; Wang, Lirong; Feng, Huanqing; Yao, Xuebiao

    2006-03-20

    As a reversible and dynamic post-translational modification (PTM) of proteins, phosphorylation plays essential regulatory roles in a broad spectrum of the biological processes. Although many studies have been contributed on the molecular mechanism of phosphorylation dynamics, the intrinsic feature of substrates specificity is still elusive and remains to be delineated. In this work, we present a novel, versatile and comprehensive program, PPSP (Prediction of PK-specific Phosphorylation site), deployed with approach of Bayesian decision theory (BDT). PPSP could predict the potential phosphorylation sites accurately for approximately 70 PK (Protein Kinase) groups. Compared with four existing tools Scansite, NetPhosK, KinasePhos and GPS, PPSP is more accurate and powerful than these tools. Moreover, PPSP also provides the prediction for many novel PKs, say, TRK, mTOR, SyK and MET/RON, etc. The accuracy of these novel PKs are also satisfying. Taken together, we propose that PPSP could be a potentially powerful tool for the experimentalists who are focusing on phosphorylation substrates with their PK-specific sites identification. Moreover, the BDT strategy could also be a ubiquitous approach for PTMs, such as sumoylation and ubiquitination, etc.

  4. Specific minor groove solvation is a crucial determinant of DNA binding site recognition

    PubMed Central

    Harris, Lydia-Ann; Williams, Loren Dean; Koudelka, Gerald B.

    2014-01-01

    The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein's ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein. PMID:25429976

  5. Site specific passive acoustic detection and densities of humpback whale calls off the coast of California

    NASA Astrophysics Data System (ADS)

    Helble, Tyler Adam

    Passive acoustic monitoring of marine mammal calls is an increasingly important method for assessing population numbers, distribution, and behavior. Automated methods are needed to aid in the analyses of the recorded data. When a mammal vocalizes in the marine environment, the received signal is a filtered version of the original waveform emitted by the marine mammal. The waveform is reduced in amplitude and distorted due to propagation effects that are influenced by the bathymetry and environment. It is important to account for these effects to determine a site-specific probability of detection for marine mammal calls in a given study area. A knowledge of that probability function over a range of environmental and ocean noise conditions allows vocalization statistics from recordings of single, fixed, omnidirectional sensors to be compared across sensors and at the same sensor over time with less bias and uncertainty in the results than direct comparison of the raw statistics. This dissertation focuses on both the development of new tools needed to automatically detect humpback whale vocalizations from single-fixed omnidirectional sensors as well as the determination of the site-specific probability of detection for monitoring sites off the coast of California. Using these tools, detected humpback calls are "calibrated" for environmental properties using the site-specific probability of detection values, and presented as call densities (calls per square kilometer per time). A two-year monitoring effort using these calibrated call densities reveals important biological and ecological information on migrating humpback whales off the coast of California. Call density trends are compared between the monitoring sites and at the same monitoring site over time. Call densities also are compared to several natural and human-influenced variables including season, time of day, lunar illumination, and ocean noise. The results reveal substantial differences in call densities

  6. Site-specific effects of the nonsteroidal anti-inflammatory drug lysine clonixinate on rat brain opioid receptors.

    PubMed

    Ortí, E; Coirini, H; Pico, J C

    1999-04-01

    In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 x 10(-4) mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

  7. Wind Power Opportunities in St. Thomas, USVI: A Site-Specific Evaluation and Analysis

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

    Lantz, E.; Warren, A.; Roberts, J. O.

    This NREL technical report utilizes a development framework originated by NREL and known by the acronym SROPTTC to assist the U.S. Virgin Islands in identifying and understanding concrete opportunities for wind power development in the territory. The report covers each of the seven components of the SROPTTC framework: Site, Resource, Off-take, Permitting, Technology, Team, and Capital as they apply to wind power in the USVI and specifically to a site in Bovoni, St. Thomas. The report concludes that Bovoni peninsula is a strong candidate for utility-scale wind generation in the territory. It represents a reasonable compromise in terms of windmore » resource, distance from residences, and developable terrain. Hurricane risk and variable terrain on the peninsula and on potential equipment transport routes add technical and logistical challenges but do not appear to represent insurmountable barriers. In addition, integration of wind power into the St. Thomas power system will present operational challenges, but based on experience in other islanded power systems, there are reasonable solutions for addressing these challenges.« less

  8. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-02-01

    Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates--both antibody-drug conjugates as well as constructs for in vivo imaging--has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope

  9. USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.

    PubMed

    Uckelmann, Michael; Densham, Ruth M; Baas, Roy; Winterwerp, Herrie H K; Fish, Alexander; Sixma, Titia K; Morris, Joanna R

    2018-01-15

    BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.

  10. Global site-specific analysis of glycoprotein N-glycan processing.

    PubMed

    Cao, Liwei; Diedrich, Jolene K; Ma, Yuanhui; Wang, Nianshuang; Pauthner, Matthias; Park, Sung-Kyu Robin; Delahunty, Claire M; McLellan, Jason S; Burton, Dennis R; Yates, John R; Paulson, James C

    2018-06-01

    N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high-mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge, we developed a robust semiquantitative mass spectrometry (MS)-based method that determines the degree of glycan occupancy at each glycosite and the proportion of N-glycans processed from high-mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 μg of protein. Here, we provide a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (iv) LC-MS/MS analysis; and (v) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved, with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/postdoc with basic skills for proteomics experiments and takes ∼7 d to complete.

  11. 76 FR 19003 - Land Disposal Restrictions: Nevada and California; Site Specific Treatment Variances for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-06

    ... both a site-specific treatment variance to U.S. Ecology Nevada (USEN) located in Beatty, Nevada and... site-specific treatment variance to U.S. Ecology Nevada (USEN) located in Beatty, Nevada and withdraw... section of this document. II. Does this action apply to me? This proposal applies only to U. S. Ecology...

  12. 78 FR 45518 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-29

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  13. 78 FR 10611 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-14

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  14. 78 FR 46330 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-31

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  15. 78 FR 53135 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-28

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  16. 77 FR 28368 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-14

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  17. 78 FR 64932 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-30

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  18. 77 FR 2713 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-19

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  19. 78 FR 7767 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  20. 78 FR 25064 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-29

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  1. 77 FR 39234 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-02

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  2. 78 FR 78952 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-27

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  3. 78 FR 54460 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-04

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  4. 78 FR 22255 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-15

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  5. 78 FR 4139 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-18

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  6. 77 FR 50488 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-21

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  7. 78 FR 36543 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-18

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  8. 78 FR 17192 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-20

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  9. 78 FR 23760 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-22

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  10. 77 FR 16021 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-19

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  11. 78 FR 32640 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-31

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  12. 76 FR 70121 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-10

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  13. 78 FR 49738 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-15

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  14. 78 FR 38969 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-28

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  15. 77 FR 24694 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-25

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  16. 77 FR 16021 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-19

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  17. 77 FR 22566 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-16

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  18. 77 FR 63300 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-16

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  19. 76 FR 64329 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-18

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Portsmouth. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  20. 78 FR 28207 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-14

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  1. 78 FR 73519 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-06

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  2. Using a site-specific technical error to establish training responsiveness: a preliminary explorative study.

    PubMed

    Weatherwax, Ryan M; Harris, Nigel K; Kilding, Andrew E; Dalleck, Lance C

    2018-01-01

    Even though cardiorespiratory fitness (CRF) training elicits numerous health benefits, not all individuals have positive training responses following a structured CRF intervention. It has been suggested that the technical error (TE), a combination of biological variability and measurement error, should be used to establish specific training responsiveness criteria to gain further insight on the effectiveness of the training program. To date, most training interventions use an absolute change or a TE from previous findings, which do not take into consideration the training site and equipment used to establish training outcomes or the specific cohort being evaluated. The purpose of this investigation was to retrospectively analyze training responsiveness of two CRF training interventions using two common criteria and a site-specific TE. Sixteen men and women completed two maximal graded exercise tests and verification bouts to identify maximal oxygen consumption (VO 2 max) and establish a site-specific TE. The TE was then used to retrospectively analyze training responsiveness in comparison to commonly used criteria: percent change of >0% and >+5.6% in VO 2 max. The TE was found to be 7.7% for relative VO 2 max. χ 2 testing showed significant differences in all training criteria for each intervention and pooled data from both interventions, except between %Δ >0 and %Δ >+7.7% in one of the investigations. Training nonresponsiveness ranged from 11.5% to 34.6%. Findings from the present study support the utility of site-specific TE criterion to quantify training responsiveness. A similar methodology of establishing a site-specific and even cohort specific TE should be considered to establish when true cardiorespiratory training adaptations occur.

  3. n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites.

    PubMed

    Xu, Longhe; Matsunaga, Felipe; Xi, Jin; Li, Min; Ma, Jingyuan; Liu, Renyu

    2014-01-01

    We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (Kd = 40 μM) with a lower affinity than SDS (Kd = 2 μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.

  4. Conversion of transuranic waste to low level waste by decontamination: a site specific update

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

    Allen, R.P.; Hazelton, R.F.

    1985-09-01

    As a followup to an FY-1984 cost/benefit study, a program was conducted in FY-1985 to transfer to the relevant DOE sites the information and technology for the direct conversion of transuranic (TRU) waste to low-level waste (LLW) by decontamination. As part of this work, the economic evaluation of the various TRUW volume reduction and conversion options was updated and expanded to include site-specific factors. The results show, for the assumptions used, that size reduction, size reduction followed by decontamination, or in situ decontamination are cost effective compared with the no-processing option. The technology transfer activities included site presentations and discussionsmore » with operations and waste management personnel to identify application opportunities and site-specific considerations and constraints that could affect the implementation of TRU waste conversion principles. These discussions disclosed definite potential for the beneficial application of these principles at most of the sites, but also confirmed the existence of site-specific factors ranging from space limitations to LLW disposal restrictions that could preclude particular applications or diminish expected benefits. 8 refs., 2 figs., 4 tabs.« less

  5. Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites in C. elegans

    PubMed Central

    Ragle, James Matthew; Katzman, Sol; Akers, Taylor F.; Barberan-Soler, Sergio; Zahler, Alan M.

    2015-01-01

    Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

  6. 78 FR 26635 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-07

    ...On April 29, 2013, the Department of Energy (DOE) published a notice of open meeting announcing a meeting on May 16, 2013 of the Environmental Management Site-Specific Advisory Board, Paducah (78 FR 25064). This document makes a correction to that notice.

  7. 77 FR 31837 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-30

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. No. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  8. 77 FR 49442 - Environmental Management Site-Specific Advisory Board, Nevada

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-16

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory Committee Act (Pub. L. No. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  9. 78 FR 16260 - Environmental Management Site-Specific Advisory Board, Paducah

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-14

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. No. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  10. 77 FR 64112 - Environmental Management Site-Specific Advisory Board, Hanford

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-18

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Hanford. The Federal Advisory Committee Act (Pub. L. No. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  11. Polymeric drug delivery systems for intraoral site-specific chemoprevention of oral cancer.

    PubMed

    Desai, Kashappa Goud H

    2018-04-01

    Oral cancer is among the most prevalent cancers in the world. Moreover, it is one of the major health problems and causes of death in many regions of the world. The traditional treatment modalities include surgical removal, radiation therapy, systemic chemotherapy, or a combination of these methods. In recent decades, there has been significant interest in intraoral site-specific chemoprevention via local drug delivery using polymeric systems. Because of its easy accessibility and clear visibility, the oral mucosa is amenable for local drug delivery. A variety of polymeric systems-such as gels, tablets, films, patches, injectable systems (e.g., millicylindrical implants, microparticles, and in situ-forming depots), and nanosized carriers (e.g., polymeric nanoparticles, nanofibers, polymer-drug conjugates, polymeric micelles, nanoliposomes, nanoemulsions, and polymersomes)-have been developed and evaluated for the local delivery of natural and synthetic chemopreventive agents. The findings of in vitro, ex vivo, and in vivo studies and the positive outcome of clinical trials demonstrate that intraoral site-specific drug delivery is an attractive, highly effective and patient-friendly strategy for the management of oral cancer. Intraoral site-specific drug delivery provides unique therapeutic advantages when compared to systemic chemotherapy. Moreover, intraoral drug delivery systems are self-administrable and can be removed when needed, increasing patient compliance. This article covers important aspects and advances related to the design, development, and efficacy of polymeric systems for intraoral site-specific drug delivery. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1383-1413, 2018. © 2017 Wiley Periodicals, Inc.

  12. Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

    NASA Astrophysics Data System (ADS)

    Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

    Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

  13. Site-specific electronic structure analysis by channeling EELS and first-principles calculations.

    PubMed

    Tatsumi, Kazuyoshi; Muto, Shunsuke; Yamamoto, Yu; Ikeno, Hirokazu; Yoshioka, Satoru; Tanaka, Isao

    2006-01-01

    Site-specific electronic structures were investigated by electron energy loss spectroscopy (EELS) under electron channeling conditions. The Al-K and Mn-L(2,3) electron energy loss near-edge structure (ELNES) of, respectively, NiAl2O4 and Mn3O4 were measured. Deconvolution of the raw spectra with the instrumental resolution function restored the blunt and hidden fine features, which allowed us to interpret the experimental spectral features by comparing with theoretical spectra obtained by first-principles calculations. The present method successfully revealed the electronic structures specific to the differently coordinated cationic sites.

  14. Lens-Specific Gene Recruitment of ζ-Crystallin through Pax6, Nrl-Maf, and Brain Suppressor Sites

    PubMed Central

    Sharon-Friling, Ronit; Richardson, Jill; Sperbeck, Sally; Lee, Douglas; Rauchman, Michael; Maas, Richard; Swaroop, Anand; Wistow, Graeme

    1998-01-01

    ζ-Crystallin is a taxon-specific crystallin, an enzyme which has undergone direct gene recruitment as a structural component of the guinea pig lens through a Pax6-dependent mechanism. Tissue specificity arises through a combination of effects involving three sites in the lens promoter. The Pax6 site (ZPE) itself shows specificity for an isoform of Pax6 preferentially expressed in lens cells. High-level expression of the promoter requires a second site, identical to an αCE2 site or half Maf response element (MARE), adjacent to the Pax6 site. A promoter fragment containing Pax6 and MARE sites gives lens-preferred induction of a heterologous promoter. Complexes binding the MARE in lens nuclear extracts are antigenically related to Nrl, and cotransfection with Nrl elevates ζ-crystallin promoter activity in lens cells. A truncated ζ promoter containing Nrl-MARE and Pax6 sites has a high level of expression in lens cells in transgenic mice but is also active in the brain. Suppression of the promoter in the brain requires sequences between −498 and −385, and a site in this region forms specific complexes in brain extract. A three-level model for lens-specific Pax6-dependent expression and gene recruitment is suggested: (i) binding of a specific isoform of Pax6; (ii) augmentation of expression through binding of Nrl or a related factor; and (iii) suppression of promoter activity in the central nervous system by an upstream negative element in the brain but not in the lens. PMID:9528779

  15. 36 CFR 6.6 - Solid waste disposal sites within new additions to the National Park System.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 1 2011-07-01 2011-07-01 false Solid waste disposal sites... NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SOLID WASTE DISPOSAL SITES IN UNITS OF THE NATIONAL PARK SYSTEM § 6.6 Solid waste disposal sites within new additions to the National Park System. (a) An operator...

  16. 36 CFR 6.6 - Solid waste disposal sites within new additions to the National Park System.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Solid waste disposal sites... NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SOLID WASTE DISPOSAL SITES IN UNITS OF THE NATIONAL PARK SYSTEM § 6.6 Solid waste disposal sites within new additions to the National Park System. (a) An operator...

  17. Studies of bioactivity, conformation and pharmacokinetic profiles of site-specific PEGylated thymosin alpha 1 derivatives.

    PubMed

    Qie, Jiankun; Ma, Jinbo; Wang, Liangyou; Xu, Xiaoyu; Zheng, Jianquan; Dong, Sijian; Xie, Jianwei; Sun, Huixian; Zhou, Wenxia; Qi, Chunhui; Zhao, Xiunan; Zhang, Yongxiang; Liu, Keliang

    2007-08-01

    Site-specific mono-PEGylations were performed in different conformational regions of Thymosin alpha 1 (T alpha 1) by introducing one cysteine residue into the chosen site and coupling with thiol-specific mPEG-MAL reagent. Results demonstrated that PEGylated sites and regions influenced the conformations and pharmacokinetic profiles of the peptide greatly with following order: alpha-helix, beta-turn, random coil and terminals, but little on the immunoactivity.

  18. Targeted, Site-specific quantitation of N- and O-glycopeptides using 18O-labeling and product ion based mass spectrometry.

    PubMed

    Srikanth, Jandhyam; Agalyadevi, Rathinasamy; Babu, Ponnusamy

    2017-02-01

    The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and 18 O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

  19. Uncovering Global SUMOylation Signaling Networks in a Site-Specific Manner

    PubMed Central

    Hendriks, Ivo A.; D’Souza, Rochelle C.J.; Yang, Bing; Verlaan-de Vries, Matty; Mann, Matthias; Vertegaal, Alfred C.O.

    2014-01-01

    SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution mass spectrometry, we have studied global SUMOylation in human cells and in a site-specific manner, identifying a total of over 4,300 SUMOylation sites in over 1,600 proteins. Moreover, for the first time in excess of 1,000 SUMOylation sites were identified under standard growth conditions. SUMOylation dynamics were quantitatively studied in response to SUMO protease inhibition, proteasome inhibition and heat shock. A considerable amount of SUMOylated lysines have previously been reported to be ubiquitylated, acetylated or methylated, indicating crosstalk between SUMO and other post-translational modifications. We identified 70 phosphorylation and 4 acetylation events in close proximity to SUMOylation sites, and provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, pre-mRNA splicing and ribosome assembly. PMID:25218447

  20. A case for protein-level and site-level specificity in glycoproteomic studies of disease.

    PubMed

    Schumacher, Katherine N; Dodds, Eric D

    2016-06-01

    Abnormal glycosylation of proteins is known to be either resultant or causative of a variety of diseases. This makes glycoproteins appealing targets as potential biomarkers and focal points of molecular studies on the development and progression of human ailment. To date, a majority of efforts in disease glycoproteomics have tended to center on either determining the concentration of a given glycoprotein, or on profiling the total population of glycans released from a mixture of glycoproteins. While these approaches have demonstrated some diagnostic potential, they are inherently insensitive to the fine molecular detail which distinguishes unique and possibly disease relevant glycoforms of specific proteins. As a consequence, such analyses can be of limited sensitivity, specificity, and accuracy because they do not comprehensively consider the glycosylation status of any particular glycoprotein, or of any particular glycosylation site. Therefore, significant opportunities exist to improve glycoproteomic inquiry into disease by engaging in these studies at the level of individual glycoproteins and their exact loci of glycosylation. In this concise review, the rationale for glycoprotein and glycosylation site specificity is developed in the context of human disease glycoproteomics with an emphasis on N-glycosylation. Recent examples highlighting disease-related perturbations in glycosylation will be presented, including those involving alterations in the overall glycosylation of a specific protein, alterations in the occupancy of a given glycosylation site, and alterations in the compositional heterogeneity of glycans occurring at a given glycosylation site. Each will be discussed with particular emphasis on how protein-specific and site-specific approaches can contribute to improved discrimination between glycoproteomes and glycoproteins associated with healthy and unhealthy states.

  1. Unveiled: Interrogating the Use of Applied Drama in Multiple and Specific Sites

    ERIC Educational Resources Information Center

    Daboo, Jerri

    2007-01-01

    This article examines a view of site through postcolonial feminism to suggest that multiple and contradictory discourses of culture, location, gender and context are all vital in an understanding of a specific site when working with a community. These views are applied to a project undertaken with a group of Asian women in Britain exploring issues…

  2. Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

    PubMed

    Merkert, Sylvia; Martin, Ulrich

    2016-06-24

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

  3. Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

    PubMed

    Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

    2012-09-01

    Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  4. Nicotinic acetylcholine receptor probed with a photoactivatable agonist: improved labeling specificity by addition of CeIV/glutathione. Extension to laser flash photolabeling.

    PubMed

    Grutter, T; Goeldner, M; Kotzyba-Hibert, F

    1999-06-08

    The molecular structure of Torpedo marmorata acetylcholine binding sites has been investigated previously by photoaffinity labeling. However, besides the nicotine molecule [Middleton et al. (1991) Biochemistry 30, 6987-6997], all other photosensitive probes used for this purpose interacted only with closed receptor states. In the perspective of mapping the functional activated state, we synthesized and developed a new photoactivatable agonist of nAChR capable of alkylation of the acetylcholine (ACh) binding sites, as reported previously [Kotzyba-Hibert et al. (1997) Bioconjugate Chem. 8, 472-480]. Here, we describe the setup of experimental conditions that were made in order to optimize the photolabeling reaction and in particular its specificity. We found that subsequent addition of the oxidant ceric ion (CeIV) and reduced glutathione before the photolabeling step lowered considerably nonspecific labeling (over 90% protection with d-tubocurarine) without affecting the binding properties of the ACh binding sites. As a consequence, irradiation at 360 nm for 20 min in these new conditions gave satisfactory coupling yields (7.5%). A general mechanism was proposed to explain the successive reactions occurring and their drastic effect on the specificity of the labeling reaction. Last, these incubation conditions can be extended to nanosecond pulsed laser photolysis leading to the same specific photoincorporation as for usual irradiations (8.5% coupling yield of ACh binding sites, 77% protection with carbamylcholine). Laser flash photocoupling of a diazocyclohexadienoyl probe on nAChR was achieved for the first time. Taken together, these data indicate that future investigation of the molecular dynamics of allosteric transitions occurring at the activated ACh binding sites should be possible.

  5. Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation.

    PubMed

    Thoemmes, Stephen F; Stutzke, Crystal A; Du, Yanmei; Browning, Michael D; Buttrick, Peter M; Walker, Lori A

    2014-01-31

    Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine 150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20 μg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Evaluating Variability and Uncertainty of Geological Strength Index at a Specific Site

    NASA Astrophysics Data System (ADS)

    Wang, Yu; Aladejare, Adeyemi Emman

    2016-09-01

    Geological Strength Index (GSI) is an important parameter for estimating rock mass properties. GSI can be estimated from quantitative GSI chart, as an alternative to the direct observational method which requires vast geological experience of rock. GSI chart was developed from past observations and engineering experience, with either empiricism or some theoretical simplifications. The GSI chart thereby contains model uncertainty which arises from its development. The presence of such model uncertainty affects the GSI estimated from GSI chart at a specific site; it is, therefore, imperative to quantify and incorporate the model uncertainty during GSI estimation from the GSI chart. A major challenge for quantifying the GSI chart model uncertainty is a lack of the original datasets that have been used to develop the GSI chart, since the GSI chart was developed from past experience without referring to specific datasets. This paper intends to tackle this problem by developing a Bayesian approach for quantifying the model uncertainty in GSI chart when using it to estimate GSI at a specific site. The model uncertainty in the GSI chart and the inherent spatial variability in GSI are modeled explicitly in the Bayesian approach. The Bayesian approach generates equivalent samples of GSI from the integrated knowledge of GSI chart, prior knowledge and observation data available from site investigation. Equations are derived for the Bayesian approach, and the proposed approach is illustrated using data from a drill and blast tunnel project. The proposed approach effectively tackles the problem of how to quantify the model uncertainty that arises from using GSI chart for characterization of site-specific GSI in a transparent manner.

  7. TU-A-201-02: Treatment Site-Specific Considerations for Clinical IGRT

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

    Wijesooriya, K.

    2016-06-15

    Recent years have seen a widespread proliferation of available in-room image guidance systems for radiation therapy target localization with many centers having multiple in-room options. In this session, available imaging systems for in-room IGRT will be reviewed highlighting the main differences in workflow efficiency, targeting accuracy and image quality as it relates to target visualization. Decision-making strategies for integrating these tools into clinical image guidance protocols that are tailored to specific disease sites like H&N, lung, pelvis, and spine SBRT will be discussed. Learning Objectives: Major system characteristics of a wide range of available in-room imaging systems for IGRT. Advantagesmore » / disadvantages of different systems for site-specific IGRT considerations. Concepts of targeting accuracy and time efficiency in designing clinical imaging protocols.« less

  8. Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures*

    PubMed Central

    Jain, Kanishk; Warmack, Rebeccah A.; Stavropoulos, Peter

    2016-01-01

    In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors. PMID:27387499

  9. Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures.

    PubMed

    Jain, Kanishk; Warmack, Rebeccah A; Debler, Erik W; Hadjikyriacou, Andrea; Stavropoulos, Peter; Clarke, Steven G

    2016-08-26

    In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Position specific variation in the rate of evolution in transcription factor binding sites

    PubMed Central

    Moses, Alan M; Chiang, Derek Y; Kellis, Manolis; Lander, Eric S; Eisen, Michael B

    2003-01-01

    Background The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Results Here we analyse the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikatae to study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artefacts of computational motif finding algorithms. Conclusion As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the

  11. Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

    PubMed

    Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

    2016-02-01

    Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. © FASEB.

  12. Site-Specific Analyses for Demonstrating Compliance with 10 CFR 61 Performance Objectives - 12179

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

    Grossman, C.J.; Esh, D.W.; Yadav, P.

    2012-07-01

    The U.S. Nuclear Regulatory Commission (NRC) is proposing to amend its regulations at 10 CFR Part 61 to require low-level radioactive waste disposal facilities to conduct site-specific analyses to demonstrate compliance with the performance objectives in Subpart C. The amendments would require licensees to conduct site-specific analyses for protection of the public and inadvertent intruders as well as analyses for long-lived waste. The amendments would ensure protection of public health and safety, while providing flexibility to demonstrate compliance with the performance objectives, for current and potential future waste streams. NRC staff intends to submit proposed rule language and associated regulatorymore » basis to the Commission for its approval in early 2012. The NRC staff also intends to develop associated guidance to accompany any proposed amendments. The guidance is intended to supplement existing low-level radioactive waste guidance on issues pertinent to conducting site-specific analyses to demonstrate compliance with the performance objectives. The guidance will facilitate implementation of the proposed amendments by licensees and assist competent regulatory authorities in reviewing the site-specific analyses. Specifically, the guidance provides staff recommendations on general considerations for the site-specific analyses, modeling issues for assessments to demonstrate compliance with the performance objectives including the performance assessment, intruder assessment, stability assessment, and analyses for long-lived waste. This paper describes the technical basis for changes to the rule language and the proposed guidance associated with implementation of the rule language. The NRC staff, per Commission direction, intends to propose amendments to 10 CFR Part 61 to require licensees to conduct site-specific analyses to demonstrate compliance with performance objectives for the protection of public health and the environment. The amendments would

  13. Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

    PubMed

    Hoffmann, E; Streichert, K; Nischan, N; Seitz, C; Brunner, T; Schwagerus, S; Hackenberger, C P R; Rubini, M

    2016-05-24

    The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

  14. 78 FR 61348 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-03

    ...On September 16, 2013, in FR Doc. 2013-22453, on page 56871, the Department of Energy (DOE) published a notice of open meeting announcing a meeting on October 2, 2013 of the Environmental Management Site-Specific Advisory Board, Portsmouth (78 FR 56871). This notice announces the cancellation of this meeting.

  15. Taking to the Streets: Dutch Community Theatre Goes Site-Specific

    ERIC Educational Resources Information Center

    van Erven, Eugene

    2007-01-01

    Dutch participatory community-based theatre has thus far been largely text-based and quite apprehensive of abstract site-specific performance, which it regarded as the product of "outsider gazing" and exploitative of local residents. Quite recently, the two veteran Dutch community-based companies Stut and RWT were forced by extraordinary…

  16. Site-specific surveillance project at the Koppers Company, Inc. , National Priorities List Site, Texarkana, Texas. Final report, March 1994

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

    Not Available

    A site-specific surveillance project was conducted at the Koppers Company, a National Priorities List site. The findings indicated that living in the Koppers area was associated with a higher prevalance of reported rashes than among residents of a similar neighborhood in Texarkana not located near this site. Koppers residents did not appear to have higher rates of either adverse pregnancy outcomes or cancer than comparison residents. They did have many more concerns about their soil, water, and chemical odors in their neighborhood than did residents of the comparison neighborhood.

  17. SMART GUIDANCE AND SMARTE - TOOLS FOR DEVELOPING SITE SPECIFIC REDEVELOPMENT PLANS

    EPA Science Inventory

    Site-specific Management Approaches and Redevelopment Tools (SMART) Guidance and its electronic counterpart, SMARTe are being developed jointly with the German Federal Ministry of Education and Research and the Interstate Technology Regulatory Council. These products will assist ...

  18. Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

    PubMed

    Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

    2018-05-09

    Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  19. A Comparison of Regional and SiteSpecific Volume Estimation Equations

    Treesearch

    Joe P. McClure; Jana Anderson; Hans T. Schreuder

    1987-01-01

    Regression equations for volume by region and site class were examined for lobiolly pine. The regressions for the Coastal Plain and Piedmont regions had significantly different slopes. The results shared important practical differences in percentage of confidence intervals containing the true total volume and in percentage of estimates within a specific proportion of...

  20. A site-specific slurry application technique on grassland and on arable crops.

    PubMed

    Schellberg, Jürgen; Lock, Reiner

    2009-01-01

    There is evidence that unequal slurry application on agricultural land contributes to N losses to the environment. Heterogeneity within fields demands adequate response by means of variable rate application. A technique is presented which allows site-specific application of slurry on grassland and arable land based on pre-defined application maps. The system contains a valve controlling flow rate by an on-board PC. During operation, flow rate is measured and scaled against set point values given in the application map together with the geographic position of the site. The systems worked sufficiently precise at a flow rate between 0 and 25 l s(-1) and an offset of actual slurry flow from set point values between 0.33 and 0.67 l s(-1). Long-term experimentation is required to test if site-specific application de facto reduces N surplus within fields and so significantly contributes to the unloading of N in agricultural areas.

  1. Evaluating multicenter DTI data in Huntington's disease on site specific effects: An ex post facto approach.

    PubMed

    Müller, Hans-Peter; Grön, Georg; Sprengelmeyer, Reiner; Kassubek, Jan; Ludolph, Albert C; Hobbs, Nicola; Cole, James; Roos, Raymund A C; Duerr, Alexandra; Tabrizi, Sarah J; Landwehrmeyer, G Bernhard; Süssmuth, Sigurd D

    2013-01-01

    Assessment of the feasibility to average diffusion tensor imaging (DTI) metrics of MRI data acquired in the course of a multicenter study. Sixty-one early stage Huntington's disease patients and forty healthy controls were studied using four different MR scanners at four European sites with acquisition protocols as close as possible to a given standard protocol. The potential and feasibility of averaging data acquired at different sites was evaluated quantitatively by region-of-interest (ROI) based statistical comparisons of coefficients of variation (CV) across centers, as well as by testing for significant group-by-center differences on averaged fractional anisotropy (FA) values between patients and controls. In addition, a whole-brain based statistical between-group comparison was performed using FA maps. The ex post facto statistical evaluation of CV and FA-values in a priori defined ROIs showed no differences between sites above chance indicating that data were not systematically biased by center specific factors. Averaging FA-maps from DTI data acquired at different study sites and different MR scanner types does not appear to be systematically biased. A suitable recipe for testing on the possibility to pool multicenter DTI data is provided to permit averaging of DTI-derived metrics to differentiate patients from healthy controls at a larger scale.

  2. CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function.

    PubMed

    Washington, Shannan D; Musarrat, Farhana; Ertel, Monica K; Backes, Gregory L; Neumann, Donna M

    2018-04-15

    There are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner. IMPORTANCE The role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1. Copyright © 2018 American Society for Microbiology.

  3. Decreased Surgical Site Infection Rate in Hysterectomy: Effect of a Gynecology-Specific Bundle.

    PubMed

    Andiman, Sarah E; Xu, Xiao; Boyce, John M; Ludwig, Elizabeth M; Rillstone, Heidi R W; Desai, Vrunda B; Fan, Linda L

    2018-06-01

    We implemented a hysterectomy-specific surgical site infection prevention bundle after a higher-than-expected surgical site infection rate was identified at our institution. We evaluate how this bundle affected the surgical site infection rate, length of hospital stay, and 30-day postoperative readmission rate. This is a quality improvement study featuring retrospective analysis of a prospectively implemented, multidisciplinary team-designed surgical site infection prevention bundle that consisted of chlorhexidine-impregnated preoperative wipes, standardized aseptic surgical preparation, standardized antibiotic dosing, perioperative normothermia, surgical dressing maintenance, and direct feedback to clinicians when the protocol was breached. There were 2,099 hysterectomies completed during the 33-month study period. There were 61 surgical site infections (4.51%) in the pre-full bundle implementation period and 14 (1.87%) in the post-full bundle implementation period; we found a sustained reduction in the proportion of patients experiencing surgical site infection during the last 8 months of the study period. After adjusting for clinical characteristics, patients who underwent surgery after full implementation were less likely to develop a surgical site infection (adjusted odds ratio [OR] 0.46, P=.01) than those undergoing surgery before full implementation. Multivariable regression analysis showed no statistically significant difference in postoperative days of hospital stay (adjusted mean ratio 0.95, P=.09) or rate of readmission for surgical site infection-specific indication (adjusted OR 2.65, P=.08) between the before and after full-bundle implementation periods. The multidisciplinary implementation of a gynecologic perioperative surgical site infection prevention bundle was associated with a significant reduction in surgical site infection rate in patients undergoing hysterectomy.

  4. Analysis of the site-specific carbon isotope composition of propane by gas source isotope ratio mass spectrometer

    NASA Astrophysics Data System (ADS)

    Piasecki, Alison; Sessions, Alex; Lawson, Michael; Ferreira, A. A.; Neto, E. V. Santos; Eiler, John M.

    2016-09-01

    Site-specific isotope ratio measurements potentially provide valuable information about the formation and degradation of complex molecules-information that is lost in conventional bulk isotopic measurements. Here we discuss the background and possible applications of such measurements, and present a technique for studying the site-specific carbon isotope composition of propane at natural abundance based on mass spectrometric analysis of the intact propane molecule and its fragment ions. We demonstrate the feasibility of this approach through measurements of mixtures of natural propane and propane synthesized with site-specific 13C enrichment, and we document the limits of precision of our technique. We show that mass balance calculations of the bulk δ13C of propane based on our site-specific measurements is generally consistent with independent constraints on bulk δ13C. We further demonstrate the accuracy of the technique, and illustrate one of its simpler applications by documenting the site-specific carbon isotope signature associated with gas phase diffusion of propane, confirming that our measurements conform to the predictions of the kinetic theory of gases. This method can be applied to propane samples of moderate size (tens of micromoles) isolated from natural gases. Thus, it provides a means of studying the site-specific stable isotope systematics of propane at natural isotope abundances on sample sizes that are readily recovered from many natural environments. This method may also serve as a model for future techniques that apply high-resolution mass spectrometry to study the site-specific isotopic distributions of larger organic molecules, with potential applications to biosynthesis, forensics and other geochemical subjects.

  5. LASIC: Light Activated Site-Specific Conjugation of Native IgGs.

    PubMed

    Hui, James Z; Tamsen, Shereen; Song, Yang; Tsourkas, Andrew

    2015-08-19

    Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large.

  6. Design of Visco-Elastic Dampers for RC Frame for Site-Specific Earthquake

    NASA Astrophysics Data System (ADS)

    Kamatchi, P.; Rama Raju, K.; Ravisankar, K.; Iyer, Nagesh R.

    2016-12-01

    Number of Reinforced Concrete (RC) framed buildings have got damaged at Ahmedabad city, India located at about 240 km away from epicenter during January 2001, 7.6 moment magnitude (Mw) Bhuj earthquake. In the present study, two dimensional nonlinear time history dynamic analyses of a typical 13 storey frame assumed to be located at Ahmedabad is carried out with the rock level and surface level site-specific ground motion for scenario earthquake of Mw 7.6 from Bhuj. Artificial ground motions are generated using extended finite source stochastic model with seismological parameters reported in literature for 2001 Bhuj earthquake. Surface level ground motions are obtained for a typical soil profile of 100 m depth reported in literature through one dimensional equivalent linear wave propagation analyses. From the analyses, failure of frame is observed for surface level ground motions which indicates that, in addition to the in-adequacy of the cross sections and reinforcement of the RC members of the frame chosen, the rich energy content of the surface level ground motion near the fundamental time period of the frame has also contributed for the failure of frame. As a part of retrofitting measure, five Visco-elastic Dampers (VED) in chevron bracing are added to frame. For the frame considered in the present study, provision of VED is found to be effective to mitigate damage for the soil site considered.

  7. Conservative site-specific and single-copy transgenesis in human LINE-1 elements

    PubMed Central

    Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

    2016-01-01

    Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

  8. 78 FR 63172 - Environmental Management Site-Specific Advisory Board, Paducah; Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-23

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  9. SafetyAnalyst : software tools for safety management of specific highway sites

    DOT National Transportation Integrated Search

    2010-07-01

    SafetyAnalyst provides a set of software tools for use by state and local highway agencies for highway safety management. SafetyAnalyst can be used by highway agencies to improve their programming of site-specific highway safety improvements. SafetyA...

  10. SITE-SPECIFIC PROTOCOL FOR MEASURING SOIL RADON POTENTIALS FOR FLORIDA HOUSES

    EPA Science Inventory

    The report describes a protocol for site-specific measurement of radon potentials for Florida houses that is consistent with existing residential radon protection maps. The protocol gives further guidance on the possible need for radon-protective house construction features. In a...

  11. Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

    PubMed

    Oram, Mark; Woolston, Joelle E; Jacobson, Andrew D; Holmes, Randall K; Oram, Diana M

    2007-04-15

    In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

  12. Bacteriophage-based Vectors for Site-specific Insertion of DNA in the Chromosome of Corynebacteria

    PubMed Central

    Oram, Mark; Woolston, Joelle E.; Jacobson, Andrew D.; Holmes, Randall K.; Oram, Diana M.

    2007-01-01

    In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as β. β-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally-encoded genes, is regulated by the DtxR protein in response to Fe2+ levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the β-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae ΔdtxR strain. Additionally, strains of β-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for β, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species. PMID:17275217

  13. Enzyme Prodrug Therapy Achieves Site-Specific, Personalized Physiological Responses to the Locally Produced Nitric Oxide

    PubMed Central

    2018-01-01

    Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment. PMID:29570264

  14. Site-specific estimation of peak-streamflow frequency using generalized least-squares regression for natural basins in Texas

    USGS Publications Warehouse

    Asquith, William H.; Slade, R.M.

    1999-01-01

    The U.S. Geological Survey, in cooperation with the Texas Department of Transportation, has developed a computer program to estimate peak-streamflow frequency for ungaged sites in natural basins in Texas. Peak-streamflow frequency refers to the peak streamflows for recurrence intervals of 2, 5, 10, 25, 50, and 100 years. Peak-streamflow frequency estimates are needed by planners, managers, and design engineers for flood-plain management; for objective assessment of flood risk; for cost-effective design of roads and bridges; and also for the desin of culverts, dams, levees, and other flood-control structures. The program estimates peak-streamflow frequency using a site-specific approach and a multivariate generalized least-squares linear regression. A site-specific approach differs from a traditional regional regression approach by developing unique equations to estimate peak-streamflow frequency specifically for the ungaged site. The stations included in the regression are selected using an informal cluster analysis that compares the basin characteristics of the ungaged site to the basin characteristics of all the stations in the data base. The program provides several choices for selecting the stations. Selecting the stations using cluster analysis ensures that the stations included in the regression will have the most pertinent information about flooding characteristics of the ungaged site and therefore provide the basis for potentially improved peak-streamflow frequency estimation. An evaluation of the site-specific approach in estimating peak-streamflow frequency for gaged sites indicates that the site-specific approach is at least as accurate as a traditional regional regression approach.

  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. Hetero-site-specific X-ray pump-probe spectroscopy for femtosecond intramolecular dynamics

    PubMed Central

    Picón, A.; Lehmann, C. S.; Bostedt, C.; Rudenko, A.; Marinelli, A.; Osipov, T.; Rolles, D.; Berrah, N.; Bomme, C.; Bucher, M.; Doumy, G.; Erk, B.; Ferguson, K. R.; Gorkhover, T.; Ho, P. J.; Kanter, E. P.; Krässig, B.; Krzywinski, J.; Lutman, A. A.; March, A. M.; Moonshiram, D.; Ray, D.; Young, L.; Pratt, S. T.; Southworth, S. H.

    2016-01-01

    New capabilities at X-ray free-electron laser facilities allow the generation of two-colour femtosecond X-ray pulses, opening the possibility of performing ultrafast studies of X-ray-induced phenomena. Particularly, the experimental realization of hetero-site-specific X-ray-pump/X-ray-probe spectroscopy is of special interest, in which an X-ray pump pulse is absorbed at one site within a molecule and an X-ray probe pulse follows the X-ray-induced dynamics at another site within the same molecule. Here we show experimental evidence of a hetero-site pump-probe signal. By using two-colour 10-fs X-ray pulses, we are able to observe the femtosecond time dependence for the formation of F ions during the fragmentation of XeF2 molecules following X-ray absorption at the Xe site. PMID:27212390

  17. Hetero-site-specific X-ray pump-probe spectroscopy for femtosecond intramolecular dynamics.

    PubMed

    Picón, A; Lehmann, C S; Bostedt, C; Rudenko, A; Marinelli, A; Osipov, T; Rolles, D; Berrah, N; Bomme, C; Bucher, M; Doumy, G; Erk, B; Ferguson, K R; Gorkhover, T; Ho, P J; Kanter, E P; Krässig, B; Krzywinski, J; Lutman, A A; March, A M; Moonshiram, D; Ray, D; Young, L; Pratt, S T; Southworth, S H

    2016-05-23

    New capabilities at X-ray free-electron laser facilities allow the generation of two-colour femtosecond X-ray pulses, opening the possibility of performing ultrafast studies of X-ray-induced phenomena. Particularly, the experimental realization of hetero-site-specific X-ray-pump/X-ray-probe spectroscopy is of special interest, in which an X-ray pump pulse is absorbed at one site within a molecule and an X-ray probe pulse follows the X-ray-induced dynamics at another site within the same molecule. Here we show experimental evidence of a hetero-site pump-probe signal. By using two-colour 10-fs X-ray pulses, we are able to observe the femtosecond time dependence for the formation of F ions during the fragmentation of XeF2 molecules following X-ray absorption at the Xe site.

  18. 40 CFR Table 6 to Subpart Fff of... - Site-Specific Compliance Schedules and Increments of Progress a

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Site-Specific Compliance Schedules and... Constructed on or Before September 20, 1994 Pt. 62, Subpt. FFF, Table 6 Table 6 to Subpart FFF of Part 62—Site-Specific Compliance Schedules and Increments of Progress a Affected facilities at the following MWC sites...

  19. 40 CFR Table 6 to Subpart Fff of... - Site-Specific Compliance Schedules and Increments of Progress a

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Site-Specific Compliance Schedules and... Constructed on or Before September 20, 1994 Pt. 62, Subpt. FFF, Table 6 Table 6 to Subpart FFF of Part 62—Site-Specific Compliance Schedules and Increments of Progress a Affected facilities at the following MWC sites...

  20. Development and fabrication of patient-specific knee implant using additive manufacturing techniques

    NASA Astrophysics Data System (ADS)

    Zammit, Robert; Rochman, Arif

    2017-10-01

    Total knee replacement is the most effective treatment to relief pain and restore normal function in a diseased knee joint. The aim of this research was to develop a patient-specific knee implant which can be fabricated using additive manufacturing techniques and has reduced wear rates using a highly wear resistant materials. The proposed design was chosen based on implant requirements, such as reduction in wear rates as well as strong fixation. The patient-specific knee implant improves on conventional knee implants by modifying the articulating surfaces and bone-implant interfaces. Moreover, tribological tests of different polymeric wear couples were carried out to determine the optimal materials to use for the articulating surfaces. Finite element analysis was utilized to evaluate the stresses sustained by the proposed design. Finally, the patient-specific knee implant was successfully built using additive manufacturing techniques.

  1. phiC31 Integrase-Mediated Site-Specific Recombination in Barley

    PubMed Central

    Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

    2012-01-01

    The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity. PMID:23024817

  2. A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

    PubMed Central

    Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

    2016-01-01

    The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

  3. 12. "SITE PLAN." Test Area 1100. Specifications No. OC35973; Drawing ...

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

    12. "SITE PLAN." Test Area 1-100. Specifications No. OC359-73; Drawing No. 5841-C-1; D.O. SERIES AW1525/7 Rev. A. - Edwards Air Force Base, Air Force Rocket Propulsion Laboratory, Leuhman Ridge near Highways 58 & 395, Boron, Kern County, CA

  4. Development of Unmanned Aerial Vehicles for Site-Specific Crop Production Management

    USDA-ARS?s Scientific Manuscript database

    Unmanned Aerial Vehicles (UAV) have been developed and applied to support the practice of precision agriculture. Compared to piloted aircrafts, an Unmanned Aerial Vehicle can focus on much smaller crop fields with much lower flight altitude than regular airplanes to perform site-specific management ...

  5. Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

    PubMed

    Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

    2011-06-01

    Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

  6. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-04-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  7. Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

    PubMed

    Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

    2018-05-03

    Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

  8. Biologically Derived Nanoparticle Arrays via a Site-Specific Reconstitution of Ferritin and their Electrochemistry

    NASA Technical Reports Server (NTRS)

    Kim, Jae-Woo; Choi, Sang H.; Lillehei, Peter T.; King, Glen C.; Elliott, James R.; Chu, Sang-Hyon; Park, Yeonjoon; Watt, Gerald D.

    2004-01-01

    Nanoparticle arrays biologically derived from an electrochemically-controlled site-specific biomineralization were fabricated on a gold substrate through the immobilization process of biomolecules. The work reported herein includes the immobilization of ferritin with various surface modifications, the electrochemical biomineralization of ferritins with different inorganic cores, the fabrication of self-assembled arrays with the immobilized ferritin, and the electrochemical characterization of various core materials. Protein immobilization on the substrate is achieved by anchoring ferritins with dithiobis-N-succinimidyl propionate (DTSP). A reconstitution process of electrochemical site-specific biomineralization with a protein cage loads ferritins with different core materials such as Pt, Co, Mn, and Ni. The ferritin acts as a nano-scale template, a biocompatible cage, and a separator between the nanoparticles. The nano-sized metalcored ferritins on a gold substrate displayed a good electrochemical activity for the electron transport and storage, which is suitable for bioelectronics applications such as biofuel cell, bionanobattery, biosensors, etc. Keywords: Ferritin, immobilization, site-specific reconstitution, biomineralization, and bioelectronics

  9. Site-specific range uncertainties caused by dose calculation algorithms for proton therapy

    NASA Astrophysics Data System (ADS)

    Schuemann, J.; Dowdell, S.; Grassberger, C.; Min, C. H.; Paganetti, H.

    2014-08-01

    The purpose of this study was to assess the possibility of introducing site-specific range margins to replace current generic margins in proton therapy. Further, the goal was to study the potential of reducing margins with current analytical dose calculations methods. For this purpose we investigate the impact of complex patient geometries on the capability of analytical dose calculation algorithms to accurately predict the range of proton fields. Dose distributions predicted by an analytical pencil-beam algorithm were compared with those obtained using Monte Carlo (MC) simulations (TOPAS). A total of 508 passively scattered treatment fields were analyzed for seven disease sites (liver, prostate, breast, medulloblastoma-spine, medulloblastoma-whole brain, lung and head and neck). Voxel-by-voxel comparisons were performed on two-dimensional distal dose surfaces calculated by pencil-beam and MC algorithms to obtain the average range differences and root mean square deviation for each field for the distal position of the 90% dose level (R90) and the 50% dose level (R50). The average dose degradation of the distal falloff region, defined as the distance between the distal position of the 80% and 20% dose levels (R80-R20), was also analyzed. All ranges were calculated in water-equivalent distances. Considering total range uncertainties and uncertainties from dose calculation alone, we were able to deduce site-specific estimations. For liver, prostate and whole brain fields our results demonstrate that a reduction of currently used uncertainty margins is feasible even without introducing MC dose calculations. We recommend range margins of 2.8% + 1.2 mm for liver and prostate treatments and 3.1% + 1.2 mm for whole brain treatments, respectively. On the other hand, current margins seem to be insufficient for some breast, lung and head and neck patients, at least if used generically. If no case specific adjustments are applied, a generic margin of 6.3% + 1.2 mm would be

  10. Cancer registries in Japan: National Clinical Database and site-specific cancer registries.

    PubMed

    Anazawa, Takayuki; Miyata, Hiroaki; Gotoh, Mitsukazu

    2015-02-01

    The cancer registry is an essential part of any rational program of evidence-based cancer control. The cancer control program is required to strategize in a systematic and impartial manner and efficiently utilize limited resources. In Japan, the National Clinical Database (NCD) was launched in 2010. It is a nationwide prospective registry linked to various types of board certification systems regarding surgery. The NCD is a nationally validated database using web-based data collection software; it is risk adjusted and outcome based to improve the quality of surgical care. The NCD generalizes site-specific cancer registries by taking advantage of their excellent organizing ability. Some site-specific cancer registries, including pancreatic, breast, and liver cancer registries have already been combined with the NCD. Cooperation between the NCD and site-specific cancer registries can establish a valuable platform to develop a cancer care plan in Japan. Furthermore, the prognosis information of cancer patients arranged using population-based and hospital-based cancer registries can help in efficient data accumulation on the NCD. International collaboration between Japan and the USA has recently started and is expected to provide global benchmarking and to allow a valuable comparison of cancer treatment practices between countries using nationwide cancer registries in the future. Clinical research and evidence-based policy recommendation based on accurate data from the nationwide database may positively impact the public.

  11. Effectiveness of the addition of Lidocaine to a hemostatic, bioresorbable putty in the treatment of iliac crest donor site pain.

    PubMed

    Müller, Marc Andreas; Mehrkens, Arne; Zürcher, Roman; Vavken, Patrick; Valderrabano, Victor

    2014-12-08

    The harvest of iliac crest bone grafts (ICBG) is associated with relevant donor site pain, but may be lowered by the application of lidocaine loaded on biodegradable, hemostatic putty for sustained local analgesic release. The goal of this double-blind controlled trial was to assess the efficacy of adding lidocaine to a hemostatic putty (Orthostat ™) to treat donor site pain following harvest of ICBG in foot and ankle procedures. After ICBG harvest during a foot and ankle procedure, the resulting bone defect was either filled with Orthostat™ (n = 7) or with the same hemostatic putty loaded with lidocaine (Orthostat-L™, n = 7). During the first 72 postoperative hours, donor site and surgical site pain were managed by patient controlled morphine delivery and a peripheral nerve block. Donor site pain was periodically quantified on a Visual Analog (VAS) and a Wong Baker FACES scale. Pain scores were plotted over time to calculate the area under the curve (AUC) to quantify the overall pain experienced in specific time intervals. Orthostat-L™ significantly reduced donor site pain over the first 12 hours postoperatively as evidenced by a significant decrease of the AUC in both VAS (p = 0.0366) and Wong Baker FACES pain score plots (p = 0.0024). Cumulated morphine uses were not significantly decreased with Orthostat-L™. The addition of lidocaine to a hemostatic putty offers a significant ICBG donor site pain reduction over the first 12 postoperative hours. ClinicalTrials.gov NCT01504035. Registered January 2nd 2012.

  12. Site-specific cancer risk in the Baltic cohort of Chernobyl cleanup workers, 1986-2007.

    PubMed

    Rahu, Kaja; Hakulinen, Timo; Smailyte, Giedre; Stengrevics, Aivars; Auvinen, Anssi; Inskip, Peter D; Boice, John D; Rahu, Mati

    2013-09-01

    To assess site-specific cancer risk in the Baltic cohort of Chernobyl cleanup workers, 1986-2007. The Baltic cohort includes 17,040 men from Estonia, Latvia and Lithuania who participated in the environmental cleanup after the accident at the Chernobyl Nuclear Power Station in 1986-1991 and who were followed up for cancer incidence until the end of 2007. Cancer cases diagnosed in the cohort and in the male population of each country were identified from the respective national cancer registers. The proportional incidence ratio (PIR) with 95% confidence interval (CI) was used to estimate the site-specific cancer risk in the cohort. For comparison and as it was possible, the site-specific standardised incidence ratio (SIR) was calculated for the Estonian sub-cohort, which was not feasible for the other countries. Overall, 756 cancer cases were reported during 1986-2007. A higher proportion of thyroid cancers in relation to the male population was found (PIR=2.76; 95%CI 1.63-4.36), especially among those who started their mission shortly after the accident, in April-May 1986 (PIR=6.38; 95%CI 2.34-13.89). Also, an excess of oesophageal cancers was noted (PIR=1.52; 95% CI 1.06-2.11). No increased PIRs for leukaemia or radiation-related cancer sites combined were observed. PIRs and SIRs for the Estonian sub-cohort demonstrated the same site-specific cancer risk pattern. Consistent evidence of an increase in radiation-related cancers in the Baltic cohort was not observed with the possible exception of thyroid cancer, where conclusions are hampered by known medical examination including thyroid screening among cleanup workers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Site-specific mouth rinsing can improve oral odor by altering bacterial counts. Blind crossover clinical study.

    PubMed

    Alqumber, Mohammed A; Arafa, Khaled A

    2014-11-01

    To determine whether site-specific mouth rinsing with oral disinfectants can improve oral odor beyond the traditional panoral mouth disinfection with mouth rinses by targeting specifically oral malodor implicated anaerobic bacteria. Twenty healthy fasting subjects volunteered for a blinded prospective, descriptive correlational crossover cross-section clinical trial conducted during the month of Ramadan between July and August 2013 in Albaha province in Saudi Arabia involving the application of Listerine Cool Mint mouth rinse by either the traditional panoral rinsing method, or a site-specific disinfection method targeting the subgingival and supragingival plaque and the posterior third of the tongue dorsum, while avoiding the remaining locations within the oral cavity. The viable anaerobic and aerobic bacterial counts, volatile sulfur compounds (VSCs) levels, organoleptic assessment of oral odor, and the tongue-coating index were compared at baseline, one, 5, and 9 hours after the treatment. The site-specific disinfection method reduced the VSCs and anaerobic bacterial loads while keeping the aerobic bacterial numbers higher than the traditional panoral rinsing method. Site-specific disinfection can more effectively maintain a healthy oral cavity by predominantly disinfecting the niches of anaerobic bacteria within the oral cavity.

  14. 21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. [50...

  15. Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

    PubMed Central

    Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

    2008-01-01

    SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

  16. An Intraoperative Site-specific Bone Density Device: A Pilot Test Case.

    PubMed

    Arosio, Paolo; Moschioni, Monica; Banfi, Luca Maria; Di Stefano, Anilo Alessio

    2015-08-01

    This paper reports a case of all-on-four rehabilitation where bone density at implant sites was assessed both through preoperative computed tomographic (CT) scans and using a micromotor working as an intraoperative bone density measurement device. Implant-supported rehabilitation is a predictable treatment option for tooth replacement whose success depends on the clinician's experience, the implant characteristics and location and patient-related factors. Among the latter, bone density is a determinant for the achievement of primary implant stability and, eventually, for implant success. The ability to measure bone density at the placement site before implant insertion could be important in the clinical setting. A patient complaining of masticatory impairment was presented with a plan calling for extraction of all her compromised teeth, followed by implant rehabilitation. A week before surgery, she underwent CT examination, and the bone density on the CT scans was measured. When the implant osteotomies were created, the bone density was again measured with a micromotor endowed with an instantaneous torque-measuring system. The implant placement protocols were adapted for each implant, according to the intraoperative measurements, and the patient was rehabilitated following an all-on-four immediate loading protocol. The bone density device provided valuable information beyond that obtained from CT scans, allowing for site-specific, intraoperative assessment of bone density immediately before implant placement and an estimation of primary stability just after implant insertion. Measuring jaw-bone density could help clinicians to select implant-placement protocols and loading strategies based on site-specific bone features.

  17. Evaluating multicenter DTI data in Huntington's disease on site specific effects: An ex post facto approach☆

    PubMed Central

    Müller, Hans-Peter; Grön, Georg; Sprengelmeyer, Reiner; Kassubek, Jan; Ludolph, Albert C.; Hobbs, Nicola; Cole, James; Roos, Raymund A.C.; Duerr, Alexandra; Tabrizi, Sarah J.; Landwehrmeyer, G. Bernhard; Süssmuth, Sigurd D.

    2013-01-01

    Purpose Assessment of the feasibility to average diffusion tensor imaging (DTI) metrics of MRI data acquired in the course of a multicenter study. Materials and methods Sixty-one early stage Huntington's disease patients and forty healthy controls were studied using four different MR scanners at four European sites with acquisition protocols as close as possible to a given standard protocol. The potential and feasibility of averaging data acquired at different sites was evaluated quantitatively by region-of-interest (ROI) based statistical comparisons of coefficients of variation (CV) across centers, as well as by testing for significant group-by-center differences on averaged fractional anisotropy (FA) values between patients and controls. In addition, a whole-brain based statistical between-group comparison was performed using FA maps. Results The ex post facto statistical evaluation of CV and FA-values in a priori defined ROIs showed no differences between sites above chance indicating that data were not systematically biased by center specific factors. Conclusion Averaging FA-maps from DTI data acquired at different study sites and different MR scanner types does not appear to be systematically biased. A suitable recipe for testing on the possibility to pool multicenter DTI data is provided to permit averaging of DTI-derived metrics to differentiate patients from healthy controls at a larger scale. PMID:24179771

  18. Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation.

    PubMed

    Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A; Dawson, Philip E; Deniz, Ashok A

    2018-04-12

    Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Analysis of the site-specific integration system of the Streptomyces aureofaciens phage μ1/6.

    PubMed

    Farkašovská, Jarmila; Godány, Andrej

    2012-03-01

    The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

  20. Women with peritoneal carcinomatosis of unknown origin: Efficacy of image-guided biopsy to determine site-specific diagnosis.

    PubMed

    Hewitt, M J; Anderson, K; Hall, G D; Weston, M; Hutson, R; Wilkinson, N; Perren, T J; Lane, G; Spencer, J A

    2007-01-01

    To evaluate the use of image-guided biopsy (IGB) in routine clinical practice to obtain site-specific diagnoses in women presenting with peritoneal carcinomatosis (PC). Retrospective case study. Tertiary referral centre. A total of 149 consecutive women with PC who underwent IGB. Biopsy was performed in women considered unsuitable for primary surgery because of poor performance status or disease unlikely to be optimally debulked, with a prior history of malignancy or where there was clinicoradiological uncertainty about primary tumour site. Standard haematoxylin-eosin histological analysis was supplemented with immunohistochemistry. The rate of site-specific diagnosis. A total of 149 women underwent IGB using computed tomography or ultrasound over a 6-year period. The only complication was one rectus sheath haematoma. In 138 (93%) women, a site-specific cancer diagnosis was made on the IGB (including 111 müllerian tract, 8 gastrointestinal tract, 4 breast and 3 lymphoma); in ten women, a repeat biopsy was necessary, giving an overall failure rate of 7%. In a further six women, malignancy was confirmed but a site-specific diagnosis could not be made, and in four women, biopsy showed benign tissue. A site-specific diagnosis was obtained in 29 of the 32 women (94%) with previous malignancy, of which 18/32 (56%) showed a new primary cancer. IGB is a safe and accurate technique for providing site-specific diagnoses in women with PC in routine clinical practice, including those with a previous relevant malignancy. IGB can replace laparoscopic or open biopsy in defining primary therapeutic options. The data would suggest that the biopsy should be performed with ultrasound where feasible.

  1. Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements

    PubMed Central

    Bridgewater, Laura C.; Walker, Marlan D.; Miller, Gwen C.; Ellison, Trevor A.; Holsinger, L. Daniel; Potter, Jennifer L.; Jackson, Todd L.; Chen, Reuben K.; Winkel, Vicki L.; Zhang, Zhaoping; McKinney, Sandra; de Crombrugghe, Benoit

    2003-01-01

    Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5′ region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled. PMID:12595563

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

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

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.

    2004-10-28

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

  3. 76 FR 12349 - Environmental Management Site-Specific Advisory Board, Nevada; Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-07

    ... AGENCY: Department of Energy. ACTION: Notice of open meeting: Correction SUMMARY: On February 24, 2011... Site- Specific Advisory Board, Nevada to be held on March 9, 2011 (76 FR 10343). This document makes...: [email protected] . Corrections In the Federal Register of February 24, 2011, in FR Doc. 2011-4148, on...

  4. 27. "SITE PLAN." Specifications No. OC15775, Drawing No. AF600915, sheet ...

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

    27. "SITE PLAN." Specifications No. OC1-57-75, Drawing No. AF-60-09-15, sheet 1 of 96, D.O. Series No. AF 1394/20, Rev. B. Stamped: RECORD DRAWING - AS CONSTRUCTED. Below stamp: Contract no. 5296 Rev. B, Date: 11/17/59. Site plan of 20,000-foot track, including construction phasing notes. - Edwards Air Force Base, South Base Sled Track, Edwards Air Force Base, North of Avenue B, between 100th & 140th Streets East, Lancaster, Los Angeles County, CA

  5. 44 CFR 354.5 - Description of site-specific, plume pathway EPZ biennial exercise-related component services and...

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ..., plume pathway EPZ biennial exercise-related component services and other services. 354.5 Section 354.5... Description of site-specific, plume pathway EPZ biennial exercise-related component services and other... will assess fees on licensees include the following: (a) Site-specific, plume pathway EPZ biennial...

  6. Smoking-Associated Site-Specific Differential Methylation in Buccal Mucosa in the COPDGene Study

    PubMed Central

    Qiu, Weiliang; Carey, Vincent J.; Morrow, Jarrett; Bacherman, Helene; Foreman, Marilyn G.; Hokanson, John E.; Bowler, Russell P.; Crapo, James D.; DeMeo, Dawn L.

    2015-01-01

    DNA methylation is a complex, tissue-specific phenomenon that can reflect both endogenous factors and exogenous exposures. Buccal brushings represent an easily accessible source of DNA, which may be an appropriate surrogate tissue in the study of environmental exposures and chronic respiratory diseases. Buccal brushings were obtained from a subset of current and former smokers from the COPDGene study. Genome-wide DNA methylation data were obtained in the discovery cohort (n = 82) using the Illumina HumanMethylation450K array. Empirical Bayes methods were used to test for differential methylation by current smoking status at 468,219 autosomal CpG sites using linear models adjusted for age, sex, and race. Pyrosequencing was performed in a nonoverlapping replication cohort (n = 130). Current smokers were significantly younger than former smokers in both the discovery and replication cohorts. Seven CpG sites were associated with current smoking at a false discovery rate less than 0.05 in the discovery cohort. Six of the seven significant sites were pyrosequenced in the replication cohort; five CpG sites, including sites annotated to CYP1B1 and PARVA, were replicated. Correlations between cumulative smoke exposure and time since smoking cessation were observed in a subset of the significantly associated CpG sites. A significant correlation between reduced lung function and increased radiographic emphysema with methylation at cg02162897 (CYP1B1) was observed among female subjects. Site-specific methylation of DNA isolated from buccal mucosa is associated with exposure to cigarette smoke, and may provide insights into the mechanisms underlying differential susceptibility toward the development of smoking-related chronic respiratory diseases. PMID:25517428

  7. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.

    PubMed

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-05-13

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.

  8. Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.

    PubMed

    Shinkuma, Satoru; Guo, Zongyou; Christiano, Angela M

    2016-05-17

    Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.

  9. Weigh-in-motion (WIM) data for site-specific LRFR bridge load rating.

    DOT National Transportation Integrated Search

    2011-08-12

    The live load factors in the Load and Resistant Factor Rating (LRFR) Manual are based on load data from Ontario : thought to be representative of traffic volumes nationwide. However, in accordance with the methodology for : developing site-specific l...

  10. Site-Specific Colloidal Crystal Nucleation by Template-enhanced Particle Transport

    NASA Astrophysics Data System (ADS)

    Mishra, Chandan K.; Sood, A. K.; Ganapathy, Rajesh

    The deliberate positioning of nano- and microstructures on surfaces is often a prerequisite for fabricating functional devices. While template-assisted nucleation is a promising route to self-assemble these structures, its success hinges on particles reaching target sites prior to nucleation and for nano/microscale particles, this is hampered by their small surface mobilities. We tailored surface features, which in the presence of attractive depletion interactions not only directed micrometer-sized colloids to specific sites but also subsequently guided their growth into ordered crystalline arrays of well-defined size and symmetry. By following the nucleation kinetics with single-particle resolution, we demonstrate control over nucleation density in a growth regime that has hitherto remained inaccessible. Our findings pave the way towards realizing non-trivial surface architectures composed of complex colloids/nanoparticles as well.

  11. Chemical Understanding of the Limited Site-Specificity in Molecular Inner-Shell Photofragmentation

    DOE PAGES

    Inhester, Ludger; Oostenrijk, Bart; Patanen, Minna; ...

    2018-02-14

    In many cases fragmentation of molecules upon inner-shell ionization is very unspecific with respect to the initially localized ionization site. Often this finding is interpreted in terms of an equilibration of internal energy into vibrational degrees of freedom after Auger decay. In this paper, we investigate the X-ray photofragmentation of ethyl trifluoroacetate upon core electron ionization at environmentally distinct carbon sites using photoelectron–photoion–photoion coincidence measurements and ab initio electronic structure calculations. For all four carbon ionization sites, the Auger decay weakens the same bonds and transfers the two charges to opposite ends of the molecule, which leads to a rapidmore » dissociation into three fragments, followed by further fragmentation steps. Finally, the lack of site specificity is attributed to the character of the dicationic electronic states after Auger decay instead of a fast equilibration of internal energy.« less

  12. Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

    PubMed Central

    Harris, M E; Kazantsev, A V; Chen, J L; Pace, N R

    1997-01-01

    Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme. PMID:9174092

  13. MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN

    PubMed Central

    Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze

    2016-01-01

    A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504

  14. Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

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

    Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

    Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

  15. 78 FR 75552 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-12

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  16. 77 FR 47047 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-07

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  17. 78 FR 49739 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-15

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  18. 77 FR 2714 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-19

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  19. 78 FR 44942 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-25

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  20. 78 FR 58294 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-23

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  1. 78 FR 38305 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-26

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  2. 78 FR 30910 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-23

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  3. 77 FR 45345 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-31

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  4. 78 FR 10612 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-14

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  5. 77 FR 26273 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-03

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  6. 77 FR 39234 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-02

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  7. 78 FR 23759 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-22

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  8. 76 FR 70120 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-10

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  9. 77 FR 53192 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-31

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  10. 75 FR 82004 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-29

    ... Laboratory AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory...--Radioactive Waste Management. Public Participation: The EM SSAB, Idaho National Laboratory, welcomes the...

  11. 78 FR 12747 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-25

    ... Laboratory AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory... Management System Public Participation: The EM SSAB, Idaho National Laboratory, welcomes the attendance of...

  12. Toxicity of ammonia, cadmium, and nitrobenzene to four local fishes in the Liao River, China and the derivation of site-specific water quality criteria.

    PubMed

    Liu, Zhihong; Li, Xiaojun; Tai, Peidong; Sun, Lizong; Yuan, Honghong; Yang, Xiaonan

    2018-01-01

    Water quality criteria (WQC) are considered to be an effective management tool for protecting aquatic environments. To derive site-specific WQC for an area, local data based on local species are essential to improve the applicability of WQC derived. Due to the paucity of local fish data available for the development of site-specific WQC for the Liao River, China, four local and widespread fishes (Pseudorasbora parva, Abbottina liaoningensis, Ctenogobius giurinus, and Misgurnus anguillicaudatus) were chosen to test their sensitivities to ammonia, cadmium and nitrobenzene. These compounds are common and regularly-measured pollutants in Chinese rivers. In addition to the published data for species resident in the Liao River, site-specific WQC for the three chemicals were derived using both a log-logistic species sensitivity distribution (SSD) and the method recommended by the USEPA, in line with current best practice, which were then compared with Chinese national WQC. It was found that A. liaoningensis was the most sensitive, followed, in order, by P. parva, C. giurinus and M. anguillicaudatus was the least sensitive, and this trend was the same to all three chemicals tested. When comparing the SSD derived solely from previously-published data with that including our data on local fish, there were significant differences identified among parameters describing the SSD curves for ammonia and nitrobenzene and significant differences were detected for site-specific WQC derived for all of the three chemicals. Based on the dataset with local fish data taxa, site-specific WQC of Liao River for ammonia, cadmium, and nitrobenzene were derived to be 20.53mg/L (at a pH of 7.0 and temperature of 20°C), 3.76μg/L (at a hardness of 100mg/L CaCO 3 ), and 0.49mg/L, respectively. Using the same deriving method for each chemical, the national Chinese WQC were higher than site-specific WQC derived in this study for ammonia (national WQC of 25.16mg/L) and nitrobenzene (national WQC

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

    PubMed

    Kwon, Inchan; Choi, Eun Sil

    2016-01-01

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

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

    PubMed Central

    Kwon, Inchan; Choi, Eun Sil

    2016-01-01

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

  15. Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP.

    PubMed

    Luo, Lin; Tong, Samuel J; Wall, Adam A; Khromykh, Tatiana; Sweet, Matthew J; Stow, Jennifer L

    2017-07-01

    Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site-specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull-downs from macrophage extracts. CRISPR-mediated SCIMP-deficient RAW264.7 macrophage-like cells were reconstituted with various phosphorylation-deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4-induced, site-specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk-SH2 pull-downs to identify additional known and new Csk targets in LPS-activated macrophages reveals the wider utility of our SH2 probes.

  16. Human-Specific Histone Methylation Signatures at Transcription Start Sites in Prefrontal Neurons

    PubMed Central

    Cheung, Iris; Bharadwaj, Rahul; Chou, Hsin-Jung; Houston, Isaac B.; Peter, Cyril J.; Mitchell, Amanda C.; Yao, Wei-Dong; Myers, Richard H.; Chen, Jiang-fan; Preuss, Todd M.; Rogaev, Evgeny I.; Jensen, Jeffrey D.; Weng, Zhiping; Akbarian, Schahram

    2012-01-01

    Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5–1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans. PMID:23185133

  17. Additive manufacturing of patient-specific tubular continuum manipulators

    NASA Astrophysics Data System (ADS)

    Amanov, Ernar; Nguyen, Thien-Dang; Burgner-Kahrs, Jessica

    2015-03-01

    Tubular continuum robots, which are composed of multiple concentric, precurved, elastic tubes, provide more dexterity than traditional surgical instruments at the same diameter. The tubes can be precurved such that the resulting manipulator fulfills surgical task requirements. Up to now the only material used for the component tubes of those manipulators is NiTi, a super-elastic shape-memory alloy of nickel and titan. NiTi is a cost-intensive material and fabrication processes are complex, requiring (proprietary) technology, e.g. for shape setting. In this paper, we evaluate component tubes made of 3 different thermoplastic materials (PLA, PCL and nylon) using fused filament fabrication technology (3D printing). This enables quick and cost-effective production of custom, patient-specific continuum manipulators, produced on site on demand. Stress-strain and deformation characteristics are evaluated experimentally for 16 fabricated tubes of each thermoplastic with diameters and shapes equivalent to those of NiTi tubes. Tubes made of PCL and nylon exhibit properties comparable to those made of NiTi. We further demonstrate a tubular continuum manipulator composed of 3 nylon tubes in a transnasal, transsphenoidal skull base surgery scenario in vitro.

  18. Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

    PubMed Central

    Dupont, L; Boizet-Bonhoure, B; Coddeville, M; Auvray, F; Ritzenthaler, P

    1995-01-01

    Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present. PMID:7836291

  19. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    PubMed

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-08

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  20. Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

    PubMed

    Middleton, Adam J; Wright, Joshua D; Day, Catherine L

    2017-11-10

    Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Site-specific management of cotton root rot using airborne and satellite imagery

    USDA-ARS?s Scientific Manuscript database

    Cotton root rot is a serious cotton disease that can now be effectively controlled with Topguard Terra Fungicide. The objectives of this research were to demonstrate how site-specific fungicide application could be implemented based on historical remote sensing imagery and variable rate technology. ...

  2. Use of GIS-based Site-specific Nitrogen Management for Improving Energy Efficiency

    USDA-ARS?s Scientific Manuscript database

    To our knowledge, geographical information system (GIS)-based site-specific nitrogen management (SSNM) techniques have not been used to assess agricultural energy costs and efficiency. This chapter uses SSNM case studies for corn (Zea mays L.) grown in Missouri and cotton (Gossypium hirsutum L.) gro...

  3. 75 FR 24686 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-05

    ... DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Northern New Mexico... Mexico. The Federal Advisory Committee Act (Pub. L. 92- 463, 86 Stat. 770) requires that public notice of..., San Juan Pueblo, New Mexico 87566. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern New...

  4. Light-stimulated cargo release from a core–shell structured nanocomposite for site-specific delivery

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

    Cai, Yun; Ling, Li; Li, Xiaofang

    This paper reported a core–shell structured site-specific delivery system with a light switch triggered by low energy light (λ=510 nm). Its core was composed of supermagnetic Fe{sub 3}O{sub 4} nanoparticles for magnetic guiding and targeting. Its outer shell consisted of mesoporous silica molecular sieve MCM-41 which offered highly ordered hexagonal tunnels for cargo capacity. A light switch N1-(4aH-cyclopenta[1,2-b:5,4-b′]dipyridin-5(5aH)-ylidene)benzene-1, 4-diamine (CBD) was covalently grafted into these hexagonal tunnels, serving as light stimuli acceptor with loading content of 1.1 μM/g. This composite was fully characterized and confirmed by SEM, TEM, XRD patterns, N{sub 2} adsorption/desorption, thermogravimetric analysis, IR, UV–vis absorption and emissionmore » spectra. Experimental data suggested that this composite had a core as wide as 150 nm and could be magnetically guided to specific sites. Its hexagonal tunnels were as long as 180 nm. Upon light stimuli of “on” and “off” states, controllable release was observed with short release time of ~900 s (90% capacity). - Graphical abstract: A core–shell structured site-specific delivery system with a light switch triggered by yellow light was constructed. Controllable release was observed with short release time of ~900 s (90% capacity). - Highlights: • A core–shell structured site-specific delivery system was constructed. • It consisted of Fe{sub 3}O{sub 4} core and MCM-41 shell grafted with light switch. • This delivery system was triggered by low energy light. • Controllable release was observed with short release time of ~900 s.« less

  5. Streptococci Engage TLR13 on Myeloid Cells in a Site-Specific Fashion.

    PubMed

    Kolter, Julia; Feuerstein, Reinhild; Spoeri, Evelyne; Gharun, Kourosh; Elling, Roland; Trieu-Cuot, Patrick; Goldmann, Tobias; Waskow, Claudia; Chen, Zhijian J; Kirschning, Carsten J; Deshmukh, Sachin D; Henneke, Philipp

    2016-03-15

    Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to β-hemolytic streptococci. Copyright © 2016 by The American Association of Immunologists, Inc.

  6. Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

    PubMed

    Gimble, F S; Thorner, J

    1993-10-15

    The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

  7. Impairments in Site-Specific AS160 Phosphorylation and Effects of Exercise Training

    PubMed Central

    Consitt, Leslie A.; Van Meter, Jessica; Newton, Christopher A.; Collier, David N.; Dar, Moahad S.; Wojtaszewski, Jørgen F.P.; Treebak, Jonas T.; Tanner, Charles J.; Houmard, Joseph A.

    2013-01-01

    The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult life span (18–84 years of age) and if endurance- and/or strength-oriented exercise training could rescue decrements in insulin action and skeletal muscle AS160 phosphorylation. A euglycemic-hyperinsulinemic clamp and skeletal muscle biopsies were performed in 73 individuals encompassing a wide age range (18–84 years of age), and insulin-stimulated AS160 phosphorylation was determined. Decrements in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666, and phospho-Akt substrate, but not Ser-318 or Ser-751. Twelve weeks of endurance- or strength-oriented exercise training increased whole-body insulin action and reversed impairments in AS160 phosphorylation evident in insulin-resistant aged individuals. These findings suggest that a dampening of insulin-induced phosphorylation of AS160 on specific sites in skeletal muscle contributes to the insulin resistance evident in a sedentary aging population and that exercise training is an effective intervention for treating these impairments. PMID:23801578

  8. A neural network based methodology to predict site-specific spectral acceleration values

    NASA Astrophysics Data System (ADS)

    Kamatchi, P.; Rajasankar, J.; Ramana, G. V.; Nagpal, A. K.

    2010-12-01

    A general neural network based methodology that has the potential to replace the computationally-intensive site-specific seismic analysis of structures is proposed in this paper. The basic framework of the methodology consists of a feed forward back propagation neural network algorithm with one hidden layer to represent the seismic potential of a region and soil amplification effects. The methodology is implemented and verified with parameters corresponding to Delhi city in India. For this purpose, strong ground motions are generated at bedrock level for a chosen site in Delhi due to earthquakes considered to originate from the central seismic gap of the Himalayan belt using necessary geological as well as geotechnical data. Surface level ground motions and corresponding site-specific response spectra are obtained by using a one-dimensional equivalent linear wave propagation model. Spectral acceleration values are considered as a target parameter to verify the performance of the methodology. Numerical studies carried out to validate the proposed methodology show that the errors in predicted spectral acceleration values are within acceptable limits for design purposes. The methodology is general in the sense that it can be applied to other seismically vulnerable regions and also can be updated by including more parameters depending on the state-of-the-art in the subject.

  9. Safe genetic modification of cardiac stem cells using a site-specific integration technique.

    PubMed

    Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

    2012-09-11

    Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

  10. Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

    PubMed

    Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

    2013-12-01

    Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

  11. Drought Prediction Site Specific and Regional up to Three Years in Advance

    NASA Astrophysics Data System (ADS)

    Suhler, G.; O'Brien, D. P.

    2002-12-01

    Dynamic Predictables has developed proprietary software that analyzes and predicts future climatic behavior based on past data. The programs employ both a regional thermodynamic model together with a unique predictive algorithm to achieve a high degree of prediction accuracy up to 36 months. The thermodynamic model was developed initially to explain the results of a study on global circulation models done at SUNY-Stony Brook by S. Hameed, R.G. Currie, and H. LaGrone (Int. Jour. Climatology, 15, pp.852-871, 1995). The authors pointed out that on a time scale of 2-70 months the spectrum of sea level pressure is dominated by the harmonics and subharmonics of the seasonal cycle and their combination tones. These oscillations are fundamental to an understanding of climatic variations on a sub-regional to continental basis. The oscillatory nature of these variations allows them to be used as broad based climate predictors. In addition, they can be subtracted from the data to yield residuals. The residuals are then analyzed to determine components that are predictable. The program then combines both the thermodynamic model results (the primary predictive model) with those from the residual data (the secondary model) to yield an estimate of the future behavior of the climatic variable. Spatial resolution is site specific or aggregated regional based upon appropriate length (45 years or more monthly data) and reasonable quality weather observation records. Most climate analysis has been based on monthly time-step data, but time scales on the order of days can be used. Oregon Climate Division 1 (Coastal) precipitation provides an example relating DynaPred's method to nature's observed elements in the early 2000s. The prediction's leading dynamic factors are the strong seasonal in the primary model combined with high secondary model contributions from planet Earth's Chandler Wobble (near 15 months) and what has been called the Quasi-Triennial Oscillation (QTO, near 36 months

  12. Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

    PubMed Central

    Huang, Weiyan; Zhao, Mei; Wei, Na; Wang, Xiaoxia; Cao, Huqing; Du, Quan; Liang, Zicai

    2014-01-01

    Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10−5 mg/ml- 10−1 mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types. PMID:24805924

  13. The Determination and Analysis of Site-Specific Rates of Mitochondrial Reactive Oxygen Species Production

    PubMed Central

    Quinlan, Casey L.; Perevoschikova, Irina V.; Goncalves, Renata L.S.; Hey-Mogensen, Martin; Brand, Martin D.

    2014-01-01

    Mitochondrial reactive oxygen species (ROS) are widely implicated in physiological and pathological pathways. We propose that it is critical to understand the specific sites of mitochondrial ROS production and their mechanisms of action. Mitochondria possess at least eight distinct sites of ROS production in the electron transport chain and matrix compartment. In this chapter, we describe the nature of the mitochondrial ROS-producing machinery and the relative capacities of each site. We provide detailed methods for the measurement of H2O2 release and the conditions under which maximal rates from each site can be achieved in intact skeletal muscle mitochondria. PMID:23791102

  14. Local nutrient regimes determine site-specific environmental triggers of cyanobacterial and microcystin variability in urban lakes

    NASA Astrophysics Data System (ADS)

    Sinang, S. C.; Reichwaldt, E. S.; Ghadouani, A.

    2014-10-01

    Toxic cyanobacterial blooms in urban lakes present serious health hazards to humans and animals and require effective management strategies. In the management of toxic cyanobacteria blooms, understanding the roles of environmental factors is crucial. To date, a range of environmental factors have been proposed as potential triggers for the spatiotemporal variability of cyanobacterial biomass and microcystins in freshwater systems. However, the environmental triggers of cyanobacteria and microcystin variability remain a subject of debate due to contrasting findings. This issue has raised the question if the environmental triggers are site-specific and unique between water bodies. In this study, we investigated the site-specificity of environmental triggers for cyanobacterial bloom and cyanotoxins dynamics. Our study suggests that cyanobacterial dominance and cyanobacterial microcystin content variability were significantly correlated to phosphorus and iron concentrations. However, the correlations between phosphorus and iron with cyanobacterial biomass and microcystin variability were not consistent between lakes, thus suggesting a site specificity of these environmental factors. The discrepancies in the correlations could be explained by differences in local nutrient concentration and the cyanobacterial community in the systems. The findings of this study suggest that identification of site-specific environmental factors under unique local conditions is an important strategy to enhance positive outcomes in cyanobacterial bloom control measures.

  15. Bifunctional chelating agent for the design and development of site specific radiopharmaceuticals and biomolecule conjugation strategy

    DOEpatents

    Katti, Kattesh V.; Prabhu, Kandikere R.; Gali, Hariprasad; Pillarsetty, Nagavara Kishore; Volkert, Wynn A.

    2003-10-21

    There is provided a method of labeling a biomolecule with a transition metal or radiometal in a site specific manner to produce a diagnostic or therapeutic pharmaceutical compound by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radio metal or a transition metal, and covalently linking the resulting metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. Also provided is a method of synthesizing the --PR.sub.2 containing biomolecules by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radiometal or a transition metal, and covalently linking the resulting radio metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. There is provided a therapeutic or diagnostic agent comprising a --PR.sub.2 containing biomolecule.

  16. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    NASA Astrophysics Data System (ADS)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-07-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  17. Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates

    PubMed Central

    Botzanowski, Thomas; Erb, Stéphane; Hernandez-Alba, Oscar; Ehkirch, Anthony; Colas, Olivier; Wagner-Rousset, Elsa; Rabuka, David; Beck, Alain; Drake, Penelope M.; Cianférani, Sarah

    2017-01-01

    ABSTRACT Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies. PMID:28406343

  18. Sequence harmony: detecting functional specificity from alignments

    PubMed Central

    Feenstra, K. Anton; Pirovano, Walter; Krab, Klaas; Heringa, Jaap

    2007-01-01

    Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww. PMID:17584793

  19. Additional disturbances as a beneficial tool for restoration of post-mining sites: a multi-taxa approach.

    PubMed

    Řehounková, Klára; Čížek, Lukáš; Řehounek, Jiří; Šebelíková, Lenka; Tropek, Robert; Lencová, Kamila; Bogusch, Petr; Marhoul, Pavel; Máca, Jan

    2016-07-01

    Open interior sands represent a highly threatened habitat in Europe. In recent times, their associated organisms have often found secondary refuges outside their natural habitats, mainly in sand pits. We investigated the effects of different restoration approaches, i.e. spontaneous succession without additional disturbances, spontaneous succession with additional disturbances caused by recreational activities, and forestry reclamation, on the diversity and conservation values of spiders, beetles, flies, bees and wasps, orthopterans and vascular plants in a large sand pit in the Czech Republic, Central Europe. Out of 406 species recorded in total, 112 were classified as open sand specialists and 71 as threatened. The sites restored through spontaneous succession with additional disturbances hosted the largest proportion of open sand specialists and threatened species. The forestry reclamations, in contrast, hosted few such species. The sites with spontaneous succession without disturbances represent a transition between these two approaches. While restoration through spontaneous succession favours biodiversity in contrast to forestry reclamation, additional disturbances are necessary to maintain early successional habitats essential for threatened species and open sand specialists. Therefore, recreational activities seem to be an economically efficient restoration tool that will also benefit biodiversity in sand pits.

  20. Site-Specific Reference Person Parameters and Derived Concentration Standards for the Savannah River Site

    DOE PAGES

    Stone, Daniel K.; Higley, Kathryn A.; Jannik, G. Timothy

    2014-05-01

    The U.S. Department of Energy Order 458.1 states that the compliance with the 1 mSv annual dose constraint to a member of the public may be demonstrated by calculating dose to the maximally exposed individual (MEI) or to a representative person. Historically, the MEI concept was used for dose compliance at the Savannah River Site (SRS) using adult dose coefficients and adult male usage parameters. For future compliance, SRS plans to use the representative person concept for dose estimates to members of the public. The representative person dose will be based on the reference person dose coefficients from the U.S.more » DOE Derived Concentration Technical Standard and on usage parameters specific to SRS for the reference and typical person. Usage parameters and dose coefficients were determined for inhalation, ingestion and external exposure pathways. The parameters for the representative person were used to calculate and tabulate SRS-specific derived concentration standards (DCSs) for the pathways not included in DOE-STD-1196-2011.« less

  1. MusiteDeep: a deep-learning framework for general and kinase-specific phosphorylation site prediction.

    PubMed

    Wang, Duolin; Zeng, Shuai; Xu, Chunhui; Qiu, Wangren; Liang, Yanchun; Joshi, Trupti; Xu, Dong

    2017-12-15

    Computational methods for phosphorylation site prediction play important roles in protein function studies and experimental design. Most existing methods are based on feature extraction, which may result in incomplete or biased features. Deep learning as the cutting-edge machine learning method has the ability to automatically discover complex representations of phosphorylation patterns from the raw sequences, and hence it provides a powerful tool for improvement of phosphorylation site prediction. We present MusiteDeep, the first deep-learning framework for predicting general and kinase-specific phosphorylation sites. MusiteDeep takes raw sequence data as input and uses convolutional neural networks with a novel two-dimensional attention mechanism. It achieves over a 50% relative improvement in the area under the precision-recall curve in general phosphorylation site prediction and obtains competitive results in kinase-specific prediction compared to other well-known tools on the benchmark data. MusiteDeep is provided as an open-source tool available at https://github.com/duolinwang/MusiteDeep. xudong@missouri.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  2. IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites.

    PubMed

    Kang, WonKyung; Imai, Noriko; Kawasaki, Yu; Nagamine, Toshihiro; Matsumoto, Shogo

    2005-11-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1-110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1-78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.

  3. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    PubMed

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  4. Site-specific investigations on aquifer thermal energy storage for space and process cooling

    NASA Astrophysics Data System (ADS)

    Brown, D. R.

    1991-08-01

    The Pacific Northwest Laboratory (PNL) has completed three preliminary site-specific feasibility studies that investigated aquifer thermal energy storage (ATES) for reducing space and process cooling costs. Chilled water stored in an ATES system could be used to meet all or part of the process and/or space cooling loads at the three facilities investigated. Seasonal or diurnal chill ATES systems could be significantly less expensive than a conventional electrically-driven, load-following chiller system at one of the three sites, depending on the cooling water loop return temperature and presumed future electricity escalation rate. For the other two sites investigated, a chill ATES system would be economically competitive with conventional chillers if onsite aquifer characteristics were improved. Well flow rates at one of the sites were adequate, but the expected thermal recovery efficiency was too low. The reverse of this situation was found at the other site, where the thermal recovery efficiency was expected to be adequate, but well flow rates were too low.

  5. Telomere formation on macronuclear chromosomes of Oxytricha trifallax and O. fallax: alternatively processed regions have multiple telomere addition sites

    PubMed Central

    Williams, Kevin R; Doak, Thomas G; Herrick, Glenn

    2002-01-01

    Background Ciliates employ massive chromatid breakage and de novo telomere formation during generation of the somatic macronucleus. Positions flanking the 81-MAC locus are reproducibly cut. But those flanking the Common Region are proposed to often escape cutting, generating three nested macronuclear chromosomes, two retaining "arms" still appended to the Common Region. Arm-distal positions must differ (in cis) from the Common Region flanks. Results The Common-Region-flanking positions also differ from the arm-distal positions in that they are "multi-TAS" regions: anchored PCR shows heterogeneous patterns of telomere addition sites, but arm-distal sites do not. The multi-TAS patterns are reproducible, but are sensitive to the sequence of the allele being processed. Thus, random degradation following chromatid cutting does not create this heterogeneity; these telomere addition sites also must be dictated by cis-acting sequences. Conclusions Most ciliates show such micro-heterogeneity in the precise positions of telomere addition sites. Telomerase is believed to be tightly associated with, and act in concert with, the chromatid-cutting nuclease: heterogeneity must be the result of intervening erosion activity. Our "weak-sites" hypothesis explains the correlation between alternative chromatid cutting at the Common Region boundaries and their multi-TAS character: when the chromatid-breakage machine encounters either a weak binding site or a weak cut site at these regions, then telomerase dissociates prematurely, leaving the new end subject to erosion by an exonuclease, which pauses at cis-acting sequences; telomerase eventually heals these resected termini. Finally, we observe TAS positioning influenced by trans-allelic interactions, reminiscent of transvection. PMID:12199911

  6. Site-specific cancer risk in the Baltic cohort of Chernobyl cleanup workers, 1986–2007

    PubMed Central

    Rahu, Kaja; Hakulinen, Timo; Smailyte, Giedre; Stengrevics, Aivars; Auvinen, Anssi; Inskip, Peter D.; Boice, John D.; Rahu, Mati

    2013-01-01

    Objective To assess site-specific cancer risk in the Baltic cohort of Chernobyl cleanup workers 1986–2007. Methods The Baltic cohort includes 17,040 men from Estonia, Latvia and Lithuania who participated in the environmental cleanup after the accident at the Chernobyl Nuclear Power Station in 1986–1991, and who were followed for cancer incidence until the end of 2007. Cancer cases diagnosed in the cohort and in the male population of each country were identified from the respective national cancer registers. The proportional incidence ratio (PIR) with 95% confidence interval (CI) was used to estimate the site-specific cancer risk in the cohort. For comparison and as it was possible, the site-specific standardized incidence ratio (SIR) was calculated for the Estonian sub-cohort, which was not feasible for the other countries. Results Overall, 756 cancer cases were reported during 1986–2007. A higher proportion of thyroid cancers in relation to the male population was found (PIR=2.76; 95%CI 1.63–4.36), especially among those who started their mission shortly after the accident, in April–May 1986 (PIR=6.38; 95% CI 2.34–13.89). Also, an excess of oesophageal cancers was noted (PIR=1.52; 95% CI 1.06–2.11). No increased PIRs for leukaemia or radiation-related cancer sites combined were observed. PIRs and SIRs for the Estonian sub-cohort demonstrated the same site-specific cancer risk pattern. Conclusion Consistent evidence of an increase in radiation-related cancers in the Baltic cohort was not observed with the possible exception of thyroid cancer, where conclusions are hampered by known medical examination including thyroid screening among cleanup workers. PMID:23683549

  7. 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

  8. Socioeconomic position, population density and site-specific cancer mortality: A multilevel analysis of Belgian adults, 2001-2011.

    PubMed

    Hagedoorn, Paulien; Vandenheede, Hadewijch; Vanthomme, Katrien; Gadeyne, Sylvie

    2018-01-01

    Our study explores the association between individual and neighborhood socioeconomic position (SEP) and all-cancer and site-specific cancer mortality. Data on all Belgian residents are retrieved from a population-based dataset constructed from the 2001 census linked to register data on emigration and mortality for 2001-2011. The study population contains all men and women aged 40 years or older during follow-up. Individual SEP is measured using education, employment status and housing conditions. Neighborhood SEP is measured by a deprivation index (in quintiles). Directly age-standardized mortality rates and multilevel Poisson models are used to estimate the association between individual SEP and neighborhood deprivation and mortality from all-cancer and cancer of the lung, colon and rectum, pancreas, prostate and female breast. The potential confounding role of population density is assessed using multilevel models as well. Our findings show an increase in mortality from all-cancer and site-specific cancer by decreasing level of individual SEP for both men and women. In addition, individuals living in highly deprived neighborhoods experience significantly higher mortality from all-cancer, lung cancer, pancreatic cancer and female colorectal cancer after controlling for individual SEP. Male colorectal and prostate cancer and female breast cancer are not associated with neighborhood deprivation. Population density acts as a confounder for female lung cancer only. Our study indicates that deprivation at both the individual and neighborhood level is associated with all-cancer mortality and mortality from several cancer sites. More research into the role of life-style related and clinical factors is necessary to gain more insight into causal pathway. © 2017 UICC.

  9. Characterizing site specific considerations for protecting aircraft during LGS operations at W. M. Keck Observatory

    NASA Astrophysics Data System (ADS)

    Stomski, Paul J., Jr.; Campbell, Randy; McCann, Kevin; Shimko, Steve

    2010-07-01

    W. M. Keck Observatory (WMKO) routinely operates laser guide star (LGS) Adaptive Optics (AO) systems at the telescope facility on the Big Island of Hawaii. One of the operational requirements for the LGS system is that a safety system to prevent nearby aircraft from being adversely affected by the laser must be provided. We will support operations in the near term with human aircraft spotters until we can successfully develop and get the appropriate approvals needed for an Automated, Integrated and Reliable System for an Aircraft Friendly Environment (AIRSAFE). This report describes some of the preliminary requirements development work at WMKO in support of the future development of AIRSAFE. We discuss the results of recent work to characterize site specific considerations that impact requirements development. The site specific considerations include the proximity of WMKO laser operations to nearby commercial airports, the implications of military operations in the area and the character of the air traffic volume and flight patterns over the telescope facility. Finally, we discuss how the design and implementation of AIRSAFE will be impacted by these site specific considerations.

  10. Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

    PubMed Central

    Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

    2012-01-01

    Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

  11. Site-specific management of cotton root rot using historical remote sensing imagery

    USDA-ARS?s Scientific Manuscript database

    Cotton root rot can now be effectively controlled with Topguard Terra Fungicide, but site-specific application of the fungicide can greatly reduce treatment cost as only portions of the field are infested with the disease. The overall goal of this three-year project was to demonstrate how to use his...

  12. Design, development and evaluation of a tree planting-site-specific fumigant applicator

    USDA-ARS?s Scientific Manuscript database

    The goal of this research was to use recent advances in the global positioning system (GPS) and computer technology to apply just the right amount of fumigant where it is most needed (i.e., tree-planting-site-specific application) to decrease the incidence of replant disease, and achieve the environ...

  13. METHOD FOR THE MEASUREMENT OF SITE-SPECIFIC TAUTOMERIC AND ZWITTERIONIC MICROSPECIES EQUILIBRIUM CONSTANTS

    EPA Science Inventory

    We describe a method for the individual measurement of simultaneously occurring, unimolecular, site-specific “microequilibrium” constants as in, for example, prototropic tautomerism and zwitterionic equilibria. Our method represents an elaboration of that of Nygren et al. (Anal. ...

  14. In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

    PubMed Central

    Jackson, Dowdy; Atkinson, John; Guevara, Claudia I.; Zhang, Chunying; Kery, Vladimir; Moon, Sung-Ju; Virata, Cyrus; Yang, Peng; Lowe, Christine; Pinkstaff, Jason; Cho, Ho; Knudsen, Nick; Manibusan, Anthony; Tian, Feng; Sun, Ying; Lu, Yingchun; Sellers, Aaron; Jia, Xiao-Chi; Joseph, Ingrid; Anand, Banmeet; Morrison, Kendall; Pereira, Daniel S.; Stover, David

    2014-01-01

    Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs. PMID:24454709

  15. Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.

    PubMed

    Letessier, Anne; Millot, Gaël A; Koundrioukoff, Stéphane; Lachagès, Anne-Marie; Vogt, Nicolas; Hansen, R Scott; Malfoy, Bernard; Brison, Olivier; Debatisse, Michelle

    2011-02-03

    Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

  16. Site preference of ternary alloying additions to NiTi: Fe, Pt, Pd, Au, Al, Cu, Zr and Hf

    NASA Technical Reports Server (NTRS)

    Bozzolo, Guillermo; Noebe, Ronald D.; Mosca, Hugo O.

    2004-01-01

    Atomistic modeling of the site substitution behavior of Pd in NiTi (J. Alloys and Comp. (2004), in press) has been extended to examine the behavior of several other alloying additions, namely, Fe, Pt, Au, Al, Cu, Zr and Hf in this important shape memory alloy. It was found that all elements, to a varying degree, displayed absolute preference for available sites in the deficient sublattice. How- ever, the energetics of the different substitutional schemes, coupled with large scale simulations indicate that the general trend in all cases is for the ternary addition to want to form stronger ordered structures with Ti.

  17. WAG 2 remedial investigation and site investigation site-specific work plan/health and safety checklist for the soil and sediment task. Environmental Restoration Program

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

    Holt, V.L.; Burgoa, B.B.

    1993-12-01

    This document is a site-specific work plan/health and safety checklist (WP/HSC) for a task of the Waste Area Grouping 2 Remedial Investigation and Site Investigation (WAG 2 RI&SI). Title 29 CFR Part 1910.120 requires that a health and safety program plan that includes site- and task-specific information be completed to ensure conformance with health- and safety-related requirements. To meet this requirement, the health and safety program plan for each WAG 2 RI&SI field task must include (1) the general health and safety program plan for all WAG 2 RI&SI field activities and (2) a WP/HSC for that particular field task.more » These two components, along with all applicable referenced procedures, must be kept together at the work site and distributed to field personnel as required. The general health and safety program plan is the Health and Safety Plan for the Remedial Investigation and Site Investigation of Waste Area Grouping 2 at the Oak Ridge National Laboratory, Oak Ridge, Tennessee (ORNL/ER-169). The WP/HSCs are being issued as supplements to ORNL/ER-169.« less

  18. 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

  19. Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases

    PubMed Central

    Krishnakumar, Radha; Grose, Carissa; Haft, Daniel H.; Zaveri, Jayshree; Alperovich, Nina; Gibson, Daniel G.; Merryman, Chuck; Glass, John I.

    2014-01-01

    Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination. PMID:24914053

  20. Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

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

    Joshi, Rakesh; Ho, Kwok Ki; Tenney, Kristen

    2013-09-18

    Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{submore » +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.« less

  1. 78 FR 63171 - Environmental Management Site-Specific Advisory Board, Northern New Mexico; Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-23

    ...This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that public notice of this meeting be announced in the Federal Register.

  2. Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes.

    PubMed

    Minkah, Nana; Hwang, Young; Perry, Kay; Van Duyne, Gregory D; Hendrickson, Robert; Lefkowitz, Elliot J; Hannenhalli, Sridhar; Bushman, Frederic D

    2007-08-15

    Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

  3. Site-specific, adult bone benefits attributed to loading during youth: A preliminary longitudinal analysis.

    PubMed

    Scerpella, Tamara A; Bernardoni, Brittney; Wang, Sijian; Rathouz, Paul J; Li, Quefeng; Dowthwaite, Jodi N

    2016-04-01

    We examined site-specific bone development in relation to childhood and adolescent artistic gymnastics exposure, comparing up to 10years of prospectively acquired longitudinal data in 44 subjects, including 31 non-gymnasts (NON) and 13 gymnasts (GYM) who participated in gymnastics from pre-menarche to ≥1.9years post-menarche. Subjects underwent annual regional and whole-body DXA scans; indices of bone geometry and strength were calculated. Anthropometrics, physical activity, and maturity were assessed annually, coincident with DXA scans. Non-linear mixed effect models centered growth in bone outcomes at menarche and adjusted for menarcheal age, height, and non-bone fat-free mass to evaluate GYM-NON differences. A POST-QUIT variable assessed the withdrawal effect of quitting gymnastics. Curves for bone area, mass (BMC), and strength indices were higher in GYM than NON at both distal radius metaphysis and diaphysis (p<0.0001). At the femoral neck, greater GYM BMC (p<0.01), narrower GYM endosteal diameter (p<0.02), and similar periosteal width (p=0.09) yielded GYM advantages in narrow neck cortical thickness and buckling ratio (both p<0.001; lower BR indicates lower fracture risk). Lumbar spine and sub-head BMC were greater in GYM than NON (p<0.036). Following gymnastics cessation, GYM slopes increased for distal radius diaphysis parameters (p≤0.01) and for narrow neck BR (p=0.02). At the distal radius metaphysis, GYM BMC and compressive strength slopes decreased, as did slopes for lumbar spine BMC, femoral neck BMC, and narrow neck cortical thickness (p<0.02). In conclusion, advantages in bone mass, geometry, and strength at multiple skeletal sites were noted across growth and into young adulthood in girls who participated in gymnastics loading to at least 1.9years post-menarche. Following gymnastics cessation, advantages at cortical bone sites improved or stabilized, while advantages at corticocancellous sites stabilized or diminished. Additional longitudinal

  4. Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

    PubMed

    Keiderling, Timothy A

    2017-12-01

    Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

  5. Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

    PubMed

    Muraki, Michiro; Hirota, Kiyonori

    2017-07-03

    Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem. A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain. The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

  6. Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae▿ ‡

    PubMed Central

    Czurda, Stefan; Jechlinger, Wolfgang; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2010-01-01

    Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery. PMID:20562305

  7. Carbonylation of Mitochondrial Aconitase with 4-Hydroxy-2-(E)-nonenal: Localization and Relative Reactivity of Addition Sites

    PubMed Central

    Liu, Qingyuan; Simpson, David C.; Gronert, Scott

    2013-01-01

    Mass spectrometry was used to investigate the effects of exposing mitochondrial aconitase (ACO2) to the membrane lipid peroxidation product, 4-hydroxy-2-(E)-nonenal (HNE). ACO2 was selected for this study because (1) it is known to be inactivated by HNE, (2) elevated concentrations of HNE-adducted ACO2 have been associated with disease states, (3) extensive structural information is available, and (4) the iron-sulfur cluster in ACO2 offers a critical target for HNE adduction. The aim of this study was to relate the inactivation of ACO2 by HNE to structural features. Initially, western blotting and an enzyme activity assay were used to assess aggregate effects and then gel electrophoresis, in-gel digestion, and tandem mass spectrometry were used to identify HNE addition sites. HNE addition reaction rates were determined for the most significant sites using the iTRAQ approach. The most reactive sites were Cys358, Cys421, and Cys424, the three iron-sulfur cluster-coordinating cysteines, Cys99, the closest non-ligated cysteine to the cluster, and Cys565, which is located in the cleft leading to the active site. Interestingly, both enzyme activity assay and iTRAQ relative abundance plots appeared to be trending toward horizontal asymptotes, rather than completion. PMID:23518448

  8. Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

    PubMed Central

    Subramanian, Shoba; Hardt, Markus; Choe, Youngchool; Niles, Richard K.; Johansen, Eric B.; Legac, Jennifer; Gut, Jiri; Kerr, Iain D.; Craik, Charles S.; Rosenthal, Philip J.

    2009-01-01

    The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. PMID:19357776

  9. Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo.

    PubMed

    Yang, Byungseop; Lim, Sung In; Kim, Jong Chul; Tae, Giyoong; Kwon, Inchan

    2016-05-09

    Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-l-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFP-WT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG.

  10. Site-specific labeling of RNA at internal ribose hydroxyl groups: terbium-assisted deoxyribozymes at work.

    PubMed

    Büttner, Lea; Javadi-Zarnaghi, Fatemeh; Höbartner, Claudia

    2014-06-04

    A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb(3+) as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2'-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2',5'-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ~160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA.

  11. 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.

  12. The Skeletal Site-Specific Role of Connective Tissue Growth Factor in Prenatal Osteogenesis

    PubMed Central

    Lambi, Alex G.; Pankratz, Talia L.; Mundy, Christina; Gannon, Maureen; Barbe, Mary F.; Richtsmeier, Joan T.; Popoff, Steven N.

    2013-01-01

    Background Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted. Results We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype. Conclusions We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation. PMID:23073844

  13. Site-Specific Pre-Swelling-Directed Morphing Structures of Patterned Hydrogels.

    PubMed

    Wang, Zhi Jian; Hong, Wei; Wu, Zi Liang; Zheng, Qiang

    2017-12-11

    Morphing materials have promising applications in various fields, yet how to program the self-shaping process for specific configurations remains a challenge. Herein we show a versatile approach to control the buckling of individual domains and thus the outcome configurations of planar-patterned hydrogels. By photolithography, high-swelling disc gels were positioned in a non-swelling gel sheet; the swelling mismatch resulted in out-of-plain buckling of the disc gels. To locally control the buckling direction, masks with holes were used to guide site-specific swelling of the high-swelling gel under the holes, which built a transient through-thickness gradient and thus directed the buckling during the subsequent unmasked swelling process. Therefore, various configurations of an identical patterned hydrogel can be programmed by the pre-swelling step with different masks to encode the buckling directions of separate domains. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

    PubMed

    Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

    2009-12-11

    Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

  15. Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

    PubMed Central

    Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2015-01-01

    Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

  16. Heavy metal interaction for Andropogon scoparius and Rudbeckia hirta grown on soil from urban and rural sites with heavy metals additions

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

    Miles, L.J.; Parker, G.R.

    1979-10-01

    Little bluestem (Andropogon scoparius) and black-eyed Susan (Rudbeckia hirta) were grown in two soils with all combinations of Cd, Zn, Pb, and Cu at two levels each for 12 weeks. Germination and establishment were completely retarded by the addition of 1000 ..mu..g/g Zn as ZnCl/sub 2/, which was due to a salt effect. Neither Cd nor Cu additions affected germination. A slight decrease in germination was noted for Pb additions of 900 ..mu..g/g which may also be associated with a salt effect. Cadmium at 10- and 20-..mu..g/g addition rates did not affect top or root dry weight. Lead and Cumore » additions reduced shoot and root dry weight yields of Andropogon scoparius, root weights being more severely affected than shoot weights. Metal additions to the urban site soil did not reduce yields to the extent they did on the rural site soil. However, yields on the urban site soil control treatment were lower compared to those for the rural site control treatment. DTPA extraction levels of heavy metals were not well correlated to plant concentrations for comparisons between the two soils. It was concluded that DTPA soil extraction may not be acceptable for metal availability comparisons among soils of differing pH. Circumstantial evidence was found for both synergistic and antagonistic effects among the heavy metals. These were of a low level and no consistent response could be determined over species or soils.« less

  17. CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

    PubMed

    Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

    2017-06-16

    Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Using Publishers' Web Sites for Reference Collection Development.

    ERIC Educational Resources Information Center

    Holmberg, Melissa

    2000-01-01

    Analyzes the ways publishers' Web sites can be used by librarians to locate additional science and technology reference materials which fall within budget constraints while meeting the needs of the patrons. Reviews specific publishers' Web sites to compare features and show how they differ. (Author/LRW)

  19. Spatial Distribution and Site-Specific Spraying of Main Sucking Pests of Elm Trees.

    PubMed

    Karimzadeh, R; Iranipour, S

    2017-06-01

    Elm trees are important landscape trees and sucking insects weaken the elm trees and produce large amounts of honeydew. The main objectives of this study were to identify main honeydew-producing pests of elm trees and do site-specific spraying against these pests. To map the spatial distribution of the sucking pests in the large scale, the study area was divided into 40 × 40 m grids and one tree was chosen randomly from each grid (a total of 55 trees). These trees were sampled twice a year in 2011 and 2012. Each sample was a 30-cm branch terminal. Eight samples were taken from each tree in four cardinal directions and two canopy levels. The number of sucking insects and leaves of each sample were counted and recorded. Spatial analysis of the data was carried out using geostatistics. Kriging was used for producing prediction maps. Insecticide application was restricted to the regions with populations higher than threshold. To identify within-tree distribution of the honeydew-producing pests, six and four elm trees were chosen in 2011 and 2012 respectively, and sampled weekly. These trees were sampled as described previously. European elm scale (EES), Gossyparia spuria (Modeer) and two species of aphids were the dominant honeydew-producing pests. The results revealed that the effects of direction, canopy level and their interactions on insect populations were not statistically significant (P < 0.05). Site-specific spraying decreased the amount of insecticides used by ca. 20%, while satisfactory control of the sucking pests and honeydew excretion was obtained. Considering the environmental and economic benefits of site-specific spraying, it is worth doing more complementary works in this area.

  20. Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

    PubMed Central

    2015-01-01

    Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody’s antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community. PMID:25121619

  1. Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

    PubMed

    Hui, James Z; Tsourkas, Andrew

    2014-09-17

    Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

  2. New Method for Producing Significant Amounts of RNA Labeled at Specific Sites | Center for Cancer Research

    Cancer.gov

    Among biomacromolecules, RNA is the most versatile, and it plays indispensable roles in almost all aspects of biology. For example, in addition to serving as mRNAs coding for proteins, RNAs regulate gene expression, such as controlling where, when, and how efficiently a gene gets expressed, participate in RNA processing, encode the genetic information of some viruses, serve as scaffolds, and even possess enzymatic activity. To study these RNAs and their biological functions and to make use of those RNA activities for biomedical applications, researchers first need to make various types of RNA. For structural biologists incorporating modified or labeled nucleotides at specific sites in RNA molecules of interest is critical to gain structural insight into RNA functions. However, placing labeled or modified residue(s) in desired positions in a large RNA has not been possible until now.

  3. A METHOD FOR THE MEASUREMENT OF SITE-SPECIFIC TAUTOMERIC AND ZWITTERIONIC MICROSPECIES EQUILIBRIUM CONSTANTS

    EPA Science Inventory

    We describe a method for the individual measurement of simultaneously occurring, unimolecular, site-specific "microequilibrium" constants as in, for example, prototropic tautomerism and zwitterionic equilibria. Our method represents an elaboration of that of Nygren et al. (Anal. ...

  4. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

    PubMed Central

    Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-01-01

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  5. Investigating temporal field sampling strategies for site-specific calibration of three soil moisture-neutron intensity parameterisation methods

    NASA Astrophysics Data System (ADS)

    Iwema, J.; Rosolem, R.; Baatz, R.; Wagener, T.; Bogena, H. R.

    2015-07-01

    The Cosmic-Ray Neutron Sensor (CRNS) can provide soil moisture information at scales relevant to hydrometeorological modelling applications. Site-specific calibration is needed to translate CRNS neutron intensities into sensor footprint average soil moisture contents. We investigated temporal sampling strategies for calibration of three CRNS parameterisations (modified N0, HMF, and COSMIC) by assessing the effects of the number of sampling days and soil wetness conditions on the performance of the calibration results while investigating actual neutron intensity measurements, for three sites with distinct climate and land use: a semi-arid site, a temperate grassland, and a temperate forest. When calibrated with 1 year of data, both COSMIC and the modified N0 method performed better than HMF. The performance of COSMIC was remarkably good at the semi-arid site in the USA, while the N0mod performed best at the two temperate sites in Germany. The successful performance of COSMIC at all three sites can be attributed to the benefits of explicitly resolving individual soil layers (which is not accounted for in the other two parameterisations). To better calibrate these parameterisations, we recommend in situ soil sampled to be collected on more than a single day. However, little improvement is observed for sampling on more than 6 days. At the semi-arid site, the N0mod method was calibrated better under site-specific average wetness conditions, whereas HMF and COSMIC were calibrated better under drier conditions. Average soil wetness condition gave better calibration results at the two humid sites. The calibration results for the HMF method were better when calibrated with combinations of days with similar soil wetness conditions, opposed to N0mod and COSMIC, which profited from using days with distinct wetness conditions. Errors in actual neutron intensities were translated to average errors specifically to each site. At the semi-arid site, these errors were below the

  6. Site-specific cancer mortality inequalities by employment and occupational groups: a cohort study among Belgian adults, 2001-2011.

    PubMed

    Vanthomme, Katrien; Van den Borre, Laura; Vandenheede, Hadewijch; Hagedoorn, Paulien; Gadeyne, Sylvie

    2017-11-12

    This study probes into site-specific cancer mortality inequalities by employment and occupational group among Belgians, adjusted for other indicators of socioeconomic (SE) position. This cohort study is based on record linkage between the Belgian censuses of 1991 and 2001 and register data on emigration and mortality for 01/10/2001 to 31/12/2011. Belgium. The study population contains all Belgians within the economically active age (25-65 years) at the census of 1991. Both absolute and relative measures were calculated. First, age-standardised mortality rates have been calculated, directly standardised to the Belgian population. Second, mortality rate ratios were calculated using Poisson's regression, adjusted for education, housing conditions, attained age, region and migrant background. This study highlights inequalities in site-specific cancer mortality, both related to being employed or not and to the occupational group of the employed population. Unemployed men and women show consistently higher overall and site-specific cancer mortality compared with the employed group. Also within the employed group, inequalities are observed by occupational group. Generally manual workers and service and sales workers have higher site-specific cancer mortality rates compared with white-collar workers and agricultural and fishery workers. These inequalities are manifest for almost all preventable cancer sites, especially those cancer sites related to alcohol and smoking such as cancers of the lung, oesophagus and head and neck. Overall, occupational inequalities were less pronounced among women compared with men. Important SE inequalities in site-specific cancer mortality were observed by employment and occupational group. Ensuring financial security for the unemployed is a key issue in this regard. Future studies could also take a look at other working regimes, for instance temporary employment or part-time employment and their relation to health. © Article author(s) (or

  7. PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome

    PubMed Central

    2010-01-01

    Background The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe. Results In this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. The phiC31 recombinase excised the attB and attP-flanked DNA, and the excision event was detected in subsequent generations in the absence of the phiC31 gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing. Conclusion The phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 in planta demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants. PMID:20178628

  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. Using the Textpresso Site-Specific Recombinases Web server to identify Cre expressing mouse strains and floxed alleles.

    PubMed

    Condie, Brian G; Urbanski, William M

    2014-01-01

    Effective tools for searching the biomedical literature are essential for identifying reagents or mouse strains as well as for effective experimental design and informed interpretation of experimental results. We have built the Textpresso Site Specific Recombinases (Textpresso SSR) Web server to enable researchers who use mice to perform in-depth searches of a rapidly growing and complex part of the mouse literature. Our Textpresso Web server provides an interface for searching the full text of most of the peer-reviewed publications that report the characterization or use of mouse strains that express Cre or Flp recombinase. The database also contains most of the publications that describe the characterization or analysis of strains carrying conditional alleles or transgenes that can be inactivated or activated by site-specific recombinases such as Cre or Flp. Textpresso SSR complements the existing online databases that catalog Cre and Flp expression patterns by providing a unique online interface for the in-depth text mining of the site specific recombinase literature.

  10. 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

  11. Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

    PubMed Central

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

    A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

  12. Nurses in need of additional support: web sites offering information in eldercare nursing environments.

    PubMed

    Matusitz, Jonathan; Breen, Gerald-Mark; Marathe, Shriram S; Wan, Thomas T H

    2010-01-01

    Studies have shown the usefulness of telemedicine and telecare in multiple settings. One form of telemedicine is e-health. Residents of nursing homes are a unique population that may significantly benefit from the e-health resources available to their caregivers. E-health Web sites appear to be viable, feasible, and timely interventional methods to provide the additional knowledge and support practitioners in these settings may need to provide preventative, reactive, and remedial care for frail residents.

  13. A site specific model and analysis of the neutral somatic mutation rate in whole-genome cancer data.

    PubMed

    Bertl, Johanna; Guo, Qianyun; Juul, Malene; Besenbacher, Søren; Nielsen, Morten Muhlig; Hornshøj, Henrik; Pedersen, Jakob Skou; Hobolth, Asger

    2018-04-19

    Detailed modelling of the neutral mutational process in cancer cells is crucial for identifying driver mutations and understanding the mutational mechanisms that act during cancer development. The neutral mutational process is very complex: whole-genome analyses have revealed that the mutation rate differs between cancer types, between patients and along the genome depending on the genetic and epigenetic context. Therefore, methods that predict the number of different types of mutations in regions or specific genomic elements must consider local genomic explanatory variables. A major drawback of most methods is the need to average the explanatory variables across the entire region or genomic element. This procedure is particularly problematic if the explanatory variable varies dramatically in the element under consideration. To take into account the fine scale of the explanatory variables, we model the probabilities of different types of mutations for each position in the genome by multinomial logistic regression. We analyse 505 cancer genomes from 14 different cancer types and compare the performance in predicting mutation rate for both regional based models and site-specific models. We show that for 1000 randomly selected genomic positions, the site-specific model predicts the mutation rate much better than regional based models. We use a forward selection procedure to identify the most important explanatory variables. The procedure identifies site-specific conservation (phyloP), replication timing, and expression level as the best predictors for the mutation rate. Finally, our model confirms and quantifies certain well-known mutational signatures. We find that our site-specific multinomial regression model outperforms the regional based models. The possibility of including genomic variables on different scales and patient specific variables makes it a versatile framework for studying different mutational mechanisms. Our model can serve as the neutral null model

  14. A novel site-specific recombination system derived from bacteriophage phiMR11.

    PubMed

    Rashel, Mohammad; Uchiyama, Jumpei; Ujihara, Takako; Takemura, Iyo; Hoshiba, Hiroshi; Matsuzaki, Shigenobu

    2008-04-04

    We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.

  15. Prognostic value of site-specific extra-hepatic disease in hepatocellular carcinoma: a SEER database analysis.

    PubMed

    Oweira, Hani; Petrausch, Ulf; Helbling, Daniel; Schmidt, Jan; Mehrabi, Arianeb; Schöb, Othmar; Giryes, Anwar; Abdel-Rahman, Omar

    2017-07-01

    We the prognostic value of site-specific extra-hepatic disease in hepatocellular carcinoma (HCC) patients registered within the surveillance, epidemiology and end results (SEER) database. SEER database (2010-2013) has been queried through SEER*Stat program to determine the prognosis of advanced HCC patients according to the site of extra-hepatic disease. Survival analysis has been conducted through Kaplan Meier analysis. A total of 4396 patients with stage IV HCC were identified in the period from 2010-2013 and they were included into this analysis. Patients with isolated regional lymph node involvement have better outcomes compared to patients with any other site of extra-hepatic disease (P < 0.0001 for both endpoints). Among patients with distant metastases, patients with bone metastases have better outcomes compared to patients with lung metastases (P < 0.0001 for both endpoints). Multivariate analysis revealed that younger age, normal alpha fetoprotein, single site of extra-hepatic disease, local treatment to the primary tumor and surgery to the metastatic disease were associated with better overall survival and liver cancer-specific survival. Within the limits of the current SEER analysis, HCC patients with isolated lung metastases seem to have worse outcomes compared to patients with isolated bone or regional nodal metastases.​.

  16. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence.

    PubMed

    Kim, Dong Seon; Hahn, Yoonsoo

    2012-11-13

    Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  17. GSHSite: Exploiting an Iteratively Statistical Method to Identify S-Glutathionylation Sites with Substrate Specificity

    PubMed Central

    Chen, Yi-Ju; Lu, Cheng-Tsung; Huang, Kai-Yao; Wu, Hsin-Yi; Chen, Yu-Ju; Lee, Tzong-Yi

    2015-01-01

    S-glutathionylation, the covalent attachment of a glutathione (GSH) to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM) that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA). TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM) is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN) and human protein tyrosine phosphatase 1b (PTP1B). Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (http://csb.cse.yzu.edu.tw/GSHSite/), for identifying uncharacterized GSH substrate sites on the protein sequences. PMID:25849935

  18. An additional substrate binding site in a bacterial phenylalanine hydroxylase

    PubMed Central

    Ronau, Judith A.; Paul, Lake N.; Fuchs, Julian E.; Corn, Isaac R.; Wagner, Kyle T.; Liedl, Klaus R.; Abu-Omar, Mahdi M.; Das, Chittaranjan

    2014-01-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

  19. Clearing the waters: Evaluating the need for site-specific field fluorescence corrections based on turbidity measurements

    USGS Publications Warehouse

    Saraceno, John F.; Shanley, James B.; Downing, Bryan D.; Pellerin, Brian A.

    2017-01-01

    In situ fluorescent dissolved organic matter (fDOM) measurements have gained increasing popularity as a proxy for dissolved organic carbon (DOC) concentrations in streams. One challenge to accurate fDOM measurements in many streams is light attenuation due to suspended particles. Downing et al. (2012) evaluated the need for corrections to compensate for particle interference on fDOM measurements using a single sediment standard in a laboratory study. The application of those results to a large river improved unfiltered field fDOM accuracy. We tested the same correction equation in a headwater tropical stream and found that it overcompensated fDOM when turbidity exceeded ∼300 formazin nephelometric units (FNU). Therefore, we developed a site-specific, field-based fDOM correction equation through paired in situ fDOM measurements of filtered and unfiltered streamwater. The site-specific correction increased fDOM accuracy up to a turbidity as high as 700 FNU, the maximum observed in this study. The difference in performance between the laboratory-based correction equation of Downing et al. (2012) and our site-specific, field-based correction equation likely arises from differences in particle size distribution between the sediment standard used in the lab (silt) and that observed in our study (fine to medium sand), particularly during high flows. Therefore, a particle interference correction equation based on a single sediment type may not be ideal when field sediment size is significantly different. Given that field fDOM corrections for particle interference under turbid conditions are a critical component in generating accurate DOC estimates, we describe a way to develop site-specific corrections.

  20. Protein specific fluorescent microspheres for labelling a protein

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor)

    1982-01-01

    Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.

  1. Risks of all-cause and site-specific fractures among hospitalized patients with COPD

    PubMed Central

    Liao, Kuang-Ming; Liang, Fu-Wen; Li, Chung-Yi

    2016-01-01

    Abstract Patients with chronic obstructive pulmonary disease (COPD) have a high prevalence of osteoporosis. The clinical sequel of osteoporosis is fracture. Patients with COPD who experience a fracture also have increased morbidity and mortality. Currently, the types of all-cause and site-specific fracture among patients with COPD are unknown. Thus, we elucidated the all-cause and site-specific fractures among patients with COPD. A retrospective, population-based, cohort study was conducted utilizing the Taiwan Longitudinal Health Insurance Database. Patients with COPD were defined as those who were hospitalized with an International Classification of Diseases, Ninth Revision, Clinical Modification code of 490 to 492 or 496 between 2001 and 2011. The index date was set as the date of discharge. The study patients were followed from the index date to the date when they sought care for any type of fracture, date of death, date of health insurance policy termination, or the last day of 2013. The types of fracture analyzed in this study included vertebral, rib, humeral, radial and ulnar/wrist, pelvic, femoral, and tibial and fibular fractures. The cohort consisted of 11,312 patients with COPD. Among these patients, 1944 experienced fractures. The most common site-specific fractures were vertebral, femoral, rib, and forearm fractures (radius, ulna, and wrist) at 32.4%, 31%, 12%, and 11.8%, respectively. The adjusted hazard ratios of fracture were 1.71 [95% confidence interval (95% CI) = 1.56–1.87] for female patient with COPD and 1.50 (95% CI = 1.39–1.52) for patients with osteoporosis after covariate adjustment. Vertebral and hip fractures are common among patients with COPD, especially among males with COPD. Many comorbidities contribute to the high risk of fracture among patients with COPD. PMID:27749576

  2. Risks of all-cause and site-specific fractures among hospitalized patients with COPD.

    PubMed

    Liao, Kuang-Ming; Liang, Fu-Wen; Li, Chung-Yi

    2016-10-01

    Patients with chronic obstructive pulmonary disease (COPD) have a high prevalence of osteoporosis. The clinical sequel of osteoporosis is fracture. Patients with COPD who experience a fracture also have increased morbidity and mortality. Currently, the types of all-cause and site-specific fracture among patients with COPD are unknown. Thus, we elucidated the all-cause and site-specific fractures among patients with COPD.A retrospective, population-based, cohort study was conducted utilizing the Taiwan Longitudinal Health Insurance Database.Patients with COPD were defined as those who were hospitalized with an International Classification of Diseases, Ninth Revision, Clinical Modification code of 490 to 492 or 496 between 2001 and 2011. The index date was set as the date of discharge. The study patients were followed from the index date to the date when they sought care for any type of fracture, date of death, date of health insurance policy termination, or the last day of 2013. The types of fracture analyzed in this study included vertebral, rib, humeral, radial and ulnar/wrist, pelvic, femoral, and tibial and fibular fractures.The cohort consisted of 11,312 patients with COPD. Among these patients, 1944 experienced fractures. The most common site-specific fractures were vertebral, femoral, rib, and forearm fractures (radius, ulna, and wrist) at 32.4%, 31%, 12%, and 11.8%, respectively. The adjusted hazard ratios of fracture were 1.71 [95% confidence interval (95% CI) = 1.56-1.87] for female patient with COPD and 1.50 (95% CI = 1.39-1.52) for patients with osteoporosis after covariate adjustment.Vertebral and hip fractures are common among patients with COPD, especially among males with COPD. Many comorbidities contribute to the high risk of fracture among patients with COPD.

  3. Integration of aerial imaging and variable-rate technology for site-specific aerial herbicide application

    USDA-ARS?s Scientific Manuscript database

    As remote sensing and variable rate technology are becoming more available for aerial applicators, practical methodologies on effective integration of these technologies are needed for site-specific aerial applications of crop production and protection materials. The objectives of this study were to...

  4. Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

    PubMed Central

    Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

    2012-01-01

    Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747

  5. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  6. Site-Specific Mobilization of Vinyl Chloride Respiration Islands by a Mechanism Common in Dehalococcoides

    PubMed Central

    2011-01-01

    Background Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA. Results We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization. Conclusions The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes. PMID:21635780

  7. 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.

  8. Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa.

    PubMed

    Llanos, Miguel N; Ronco, Ana M; Aguirre, María C

    2003-06-27

    This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.

  9. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

    DOE PAGES

    Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; ...

    2016-02-09

    Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

  10. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

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

    Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.

    Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

  11. Bacterial Production of Site Specific 13C Labeled Phenylalanine and Methodology for High Level Incorporation into Bacterially Expressed Recombinant Proteins

    PubMed Central

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L.

    2016-01-01

    Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study. PMID:28028744

  12. Site-specific binding of a water molecule to the sulfa drugs sulfamethoxazole and sulfisoxazole: a laser-desorption isomer-specific UV and IR study.

    PubMed

    Uhlemann, Thomas; Seidel, Sebastian; Müller, Christian W

    2018-03-07

    To determine the preferred water molecule binding sites of the polybasic sulfa drugs sulfamethoxazole (SMX) and sulfisoxazole (SIX), we have studied their monomers and monohydrated complexes through laser-desorption conformer-specific UV and IR spectroscopy. Both the SMX and SIX monomer adopt a single conformer in the molecular beam. On the basis of their conformer-specific IR spectra in the NH stretch region, these conformers were assigned to the SMX and SIX global minimum structures, both exhibiting a staggered sulfonamide group and an intramolecular C-HO[double bond, length as m-dash]S hydrogen bond. The SMX-H 2 O and SIX-H 2 O complexes each adopt a single isomer in the molecular beam. Their isomeric structures were determined based on their isomer-specific IR spectra in the NH/OH stretch region. Quantum Theory of Atoms in Molecules analysis of the calculated electron densities revealed that in the SMX-H 2 O complex the water molecule donates an O-HN hydrogen bond to the heterocycle nitrogen atom and accepts an N-HO hydrogen bond from the sulfonamide NH group. In the SIX-H 2 O complex, however, the water molecule does not bind to the heterocycle but instead donates an O-HO[double bond, length as m-dash]S hydrogen bond to the sulfonamide group and accepts an N-HO hydrogen bond from the sulfonamide NH group. Both water complexes are additionally stabilized by a C ph -HOH 2 hydrogen bond. Interacting Quantum Atoms analysis suggests that all intermolecular hydrogen bonds are dominated by the short-range exchange-correlation contribution.

  13. Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium.

    PubMed

    Chen, Cui-Hua; Xu, Hu; Liu, Xun-Hong; Zou, Li-Si; Wang, Mei; Liu, Zi-Xiu; Fu, Xing-Sheng; Zhao, Hui; Yan, Ying

    2017-12-01

    Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts. © 2017 Wiley Periodicals, Inc.

  14. Integrated Decision Support, Sensor Networks and Adaptive Control for Wireless Site-specific Sprinkler Irrigation

    USDA-ARS?s Scientific Manuscript database

    The development of site-specific sprinkler irrigation water management systems will be a major factor in future efforts to improve the various efficiencies of water-use and to support a sustainable irrigated environment. The challenge is to develop fully integrated management systems with supporting...

  15. Integrated decision support, sensor networks and adaptive control for wireless site-specific sprinkler irrigation

    USDA-ARS?s Scientific Manuscript database

    The development of site-specific sprinkler irrigation water management systems will be a major factor in future efforts to improve the various efficiencies of water-use and to support a sustainable irrigated environment. The challenge is to develop fully integrated management systems with supporting...

  16. Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing.

    PubMed Central

    Hodges, P E; Navaratnam, N; Greeve, J C; Scott, J

    1991-01-01

    Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase. Images PMID:2030940

  17. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

    PubMed

    Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L; Wetsel, Rick A; Wang, Dachun

    2014-02-01

    Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of β-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration-free and exogenous reprogramming factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. © 2013 AlphaMed Press.

  19. A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells

    PubMed Central

    Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L.; Wetsel, Rick A.; Wang, Dachun

    2013-01-01

    Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector-integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types, are still major obstacles. Here we report a novel strategy using a single non-viral site-specific-targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific NeomycinR trangene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of beta-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random-integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random-integration-free and exogenous reprogramming-factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultra-structural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. PMID:24123810

  20. Site-specific recombination in the cyanobacterium Anabaena sp. strain PCC 7120 catalyzed by the integrase of coliphage HK022.

    PubMed

    Melnikov, Olga; Zaritsky, Arieh; Zarka, Aliza; Boussiba, Sammy; Malchin, Natalia; Yagil, Ezra; Kolot, Mikhail

    2009-07-01

    The integrase (Int) of the lambda-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P(glnA)-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P(glnA). The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

  1. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  2. Improving 1D Site Specific Velocity Profiles for the Kik-Net Network

    NASA Astrophysics Data System (ADS)

    Holt, James; Edwards, Benjamin; Pilz, Marco; Fäh, Donat; Rietbrock, Andreas

    2017-04-01

    Ground motion predication equations (GMPEs) form the cornerstone of modern seismic hazard assessments. When produced to a high standard they provide reliable estimates of ground motion/spectral acceleration for a given site and earthquake scenario. This information is crucial for engineers to optimise design and for regulators who enforce legal minimum safe design capacities. Classically, GMPEs were built upon the assumption that variability around the median model could be treated as aleatory. As understanding improved, it was noted that the propagation could be segregated into the response of the average path from the source and the response of the site. This is because the heterogeneity of the near-surface lithology is significantly different from that of the bulk path. It was then suggested that the semi-ergodic approach could be taken if the site response could be determined, moving uncertainty away from aleatory to epistemic. The determination of reliable site-specific response models is therefore becoming increasingly critical for ground motion models used in engineering practice. Today it is common practice to include proxies for site response within the scope of a GMPE, such as Vs30 or site classification, in an effort to reduce the overall uncertainty of the predication at a given site. However, these proxies are not always reliable enough to give confident ground motion estimates, due to the complexity of the near-surface. Other approaches of quantifying the response of the site include detailed numerical simulations (1/2/3D - linear, EQL, non-linear etc.). However, in order to be reliable, they require highly detailed and accurate velocity and, for non-linear analyses, material property models. It is possible to obtain this information through invasive methods, but is expensive, and not feasible for most projects. Here we propose an alternative method to derive reliable velocity profiles (and their uncertainty), calibrated using almost 20 years of

  3. Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

    NASA Astrophysics Data System (ADS)

    Yu, Yuan; Tong, Qi; Li, Zhongxia; Tian, Jinhai; Wang, Yizhi; Su, Feng; Wang, Yongsheng; Liu, Jun; Zhang, Yong

    2014-02-01

    PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.

  4. Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry.

    PubMed

    Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H; Petersen, Lars C; Kristensen, Jesper B; Behrens, Carsten; Madsen, Jacob; Kjaer, Andreas

    2018-01-17

    A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64 Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64 Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64 Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

  5. The phage integrase vector pIPI03 allows RecA-independent, site-specific labelling of Staphylococcus lugdunensis strains.

    PubMed

    Heilbronner, Simon; Monk, Ian R; Foster, Timothy J

    2013-11-01

    Staphylococcus lugdunensis is a coagulase negative staphylococcus that is a commensal of man and an opportunistic pathogen. A site-specific integrative plasmid for the use in S. lugdunensis was constructed and validated. The integrase gene ccrB of bacteriophage ϕSL01 together with its attachment site was cloned into the thermosensitive plasmid pIMAY. The resulting plasmid pIPI03 integrated RecA-independently, site-specifically and irreversibly into the S. lugdunensis chromosome. Two IPTG-inducible antibiotic resistance determinants were cloned into pIPI03 and the derivatives were used to construct strains suitable for competitive growth experiments in both in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    PubMed Central

    2012-01-01

    Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution. PMID:23148531

  7. Development, testing, and applications of site-specific tsunami inundation models for real-time forecasting

    NASA Astrophysics Data System (ADS)

    Tang, L.; Titov, V. V.; Chamberlin, C. D.

    2009-12-01

    The study describes the development, testing and applications of site-specific tsunami inundation models (forecast models) for use in NOAA's tsunami forecast and warning system. The model development process includes sensitivity studies of tsunami wave characteristics in the nearshore and inundation, for a range of model grid setups, resolutions and parameters. To demonstrate the process, four forecast models in Hawaii, at Hilo, Kahului, Honolulu, and Nawiliwili are described. The models were validated with fourteen historical tsunamis and compared with numerical results from reference inundation models of higher resolution. The accuracy of the modeled maximum wave height is greater than 80% when the observation is greater than 0.5 m; when the observation is below 0.5 m the error is less than 0.3 m. The error of the modeled arrival time of the first peak is within 3% of the travel time. The developed forecast models were further applied to hazard assessment from simulated magnitude 7.5, 8.2, 8.7 and 9.3 tsunamis based on subduction zone earthquakes in the Pacific. The tsunami hazard assessment study indicates that use of a seismic magnitude alone for a tsunami source assessment is inadequate to achieve such accuracy for tsunami amplitude forecasts. The forecast models apply local bathymetric and topographic information, and utilize dynamic boundary conditions from the tsunami source function database, to provide site- and event-specific coastal predictions. Only by combining a Deep-ocean Assessment and Reporting of Tsunami-constrained tsunami magnitude with site-specific high-resolution models can the forecasts completely cover the evolution of earthquake-generated tsunami waves: generation, deep ocean propagation, and coastal inundation. Wavelet analysis of the tsunami waves suggests the coastal tsunami frequency responses at different sites are dominated by the local bathymetry, yet they can be partially related to the locations of the tsunami sources. The study

  8. Coupling interactions between voltage sensors of the sodium channel as revealed by site-specific measurements.

    PubMed

    Chanda, Baron; Asamoah, Osei Kwame; Bezanilla, Francisco

    2004-03-01

    The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains. Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.

  9. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

    PubMed Central

    Ngô, V; Gourdji, D; Laverrière, J N

    1996-01-01

    The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

  10. Site-Specific Research Conducted in Support of the Salton Sea Solar Pond Project - FY 1982 Report

    NASA Technical Reports Server (NTRS)

    French, R. L.; Marsh, H. E.; Roschke, E. J.; Wu, Y. C.

    1984-01-01

    The design and operation of a salt-gradient solar pond power plant at the Salton Sea presents problems not encountered at small research ponds that were built in the United States. The specific characteristics of the Salton Sea site and the desire to construct the pond using the local clay as a sealant represent major deviations from previous solar pond experience. The site-specific research in support of the plant design is described. The research activity included validation of the spectrophotometric light transmission measurement technique, a search for options for clarifying the turbid and colored water of the Salton Sea, development of water clarification specifications in terms common to industry practice, quantification of gas production from microbiological reactions in the ground, a determination of the combined effects of temperature and salinity on the permeation of the local clays, and a preliminary evaluation of material corrosion.

  11. Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

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

    Su, Dian; Shukla, Anil K.; Chen, Baowei

    2013-04-01

    S-nitrosylation (SNO) is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. While many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge due to the low-abundance and labile nature of the modification. Herein we present further improvement and optimization of the recently reported, resin-assisted cysteinyl peptide enrichment protocol for SNO identification and the extension of this application to mouse skeletal muscle to identify specific sites sensitive to S-nitrosylation by quantitative reactivity profiling. The results of our data indicate that the protein- andmore » peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione (GSNO), an NO donor, at two different physiologically relevant concentrations (i.e., 10 μM and 100 μM). The reactivity profiling experiments overall identified 489 SNO-modified cysteine sites from 197 proteins with the specificity of 95.2% at the unique-peptide-level based on the percentage of Cys-peptides. Among these sites, 260 sites from 135 proteins were observed with relatively high reactivity to S-nitrosylation; such SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are preferentially localized in mitochondria, contractile fiber and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, the SNO-sensitive proteins seem to be primarily involved in metabolic pathways, including TCA cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism

  12. Site-Specific Antibody–Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development

    PubMed Central

    2015-01-01

    Antibody–drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering. PMID:25494884

  13. Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.

    PubMed

    Agarwal, Paresh; Bertozzi, Carolyn R

    2015-02-18

    Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering.

  14. Site-specific gene transfer into the rat spinal cord by photomechanical waves

    NASA Astrophysics Data System (ADS)

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Uozumi, Yoichi; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2011-10-01

    Nonviral, site-specific gene delivery to deep tissue is required for gene therapy of a spinal cord injury. However, an efficient method satisfying these requirements has not been established. This study demonstrates efficient and targeted gene transfer into the spinal cord by using photomechanical waves (PMWs), which were generated by irradiating a black laser absorbing rubber with 532-nm nanosecond Nd:YAG laser pulses. After a solution of plasmid DNA coding for enhanced green fluorescent protein (EGFP) or luciferase was intraparenchymally injected into the spinal cord, PMWs were applied to the target site. In the PMW application group, we observed significant EGFP gene expression in the white matter and remarkably high luciferase activity only in the spinal cord segment exposed to the PMWs. We also assessed hind limb movements 24 h after the application of PMWs based on the Basso-Beattie-Bresnahan (BBB) score to evaluate the noninvasiveness of this method. Locomotor evaluation showed no significant decrease in BBB score under optimum laser irradiation conditions. These findings demonstrated that exogenous genes can be efficiently and site-selectively delivered into the spinal cord by applying PMWs without significant locomotive damage.

  15. An Enzyme-Mediated Methodology for the Site-Specific Radiolabeling of Antibodies Based on Catalyst-Free Click Chemistry

    PubMed Central

    Zeglis, Brian M.; Davis, Charles B.; Aggeler, Robert; Kang, Hee Chol; Chen, Aimei; Agnew, Brian J.; Lewis, Jason S.

    2013-01-01

    An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal 89Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with 89Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing 89Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to high selective tumor uptake (67.5 ± 5.0 %ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic. PMID:23688208

  16. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

    PubMed

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

    2017-09-15

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

  17. Creating prescription maps from satellite imagery for site-specific management of cotton root rot

    USDA-ARS?s Scientific Manuscript database

    Cotton root rot is a century-old cotton disease that can now be controlled with Topguard Terra Fungicide. However, as this disease tends to occur in the same general areas within fields year after year, site-specific treatment can be more effective and economical. The objective of this study was to ...

  18. WAG 2 remedial investigation and site investigation site-specific work plan/health and safety checklist for the ecological assessment task, Kingfisher Study

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

    Holt, V.L.; Baron, L.A.

    1994-05-01

    This report provides specific details and requirements for the WAG 2 remedial investigation and site investigation Ecological Assessment Task, Kingfisher Study, including information that will contribute to safe completion of the project. The report includes historical background; a site map; project organization; task descriptions and hazard evaluations; controls; and monitoring, personal protective equipment, decontamination, and medical surveillance program requirements. The report also includes descriptions of site personnel and their certifications as well as suspected WAG 2 contaminants and their characteristics. The primary objective of the WAG 2 Kingfisher Study is to assess the feasibility of using kingfishers as biological monitorsmore » of contaminants on the Oak Ridge Reservation (ORR). Kingfisher sample collection will be used to determine the levels of contaminants and degree of bioaccumulation within a common piscivorous bird feeding on contaminated fish from streams on the ORR.« less

  19. Review: A Position Paper on Selenium in Ecotoxicology: A Procedure for Deriving Site-Specific Water Quality Criteria

    Treesearch

    A. Dennis Lemly

    1997-01-01

    This paper describes a method for deriving site-specific water quality criteria for selenium using a two-step process: (1) gather information on selenium residues and biological effects at the site and in down-gradient systems and (2) examine criteria based on the degree of bioaccumulation, the relationship between mea-sured residues and threshold concentrations for...

  20. Site-specific protein immobilization in a microfluidic chip channel via an IEF-gelation process.

    PubMed

    Shi, Mianhong; Peng, Youyuan; Yu, Shaoning; Liu, Baohong; Kong, Jilie

    2007-05-01

    A novel strategy for site-specific protein immobilization via combining chip IEF with low-temperature sol-gel technology, called IEF-GEL here, in the channel of a modified poly(methyl methacrylate) (PMMA) microfluidic chip is proposed in this work. The IEF-GEL process involves firstly IEF for homogeneously dissolved protein in PBS containing alumina sol and carrier ampholyte with prearranged pH gradient, and then gelation locally for protein encapsulation. The process and feasibility of proposed IEF-GEL were investigated by EOF measurements, fluorescence microscopic photography, Raman spectrum and further demonstrated by glucose oxidase (GOx) reactors integrated with end-column electrochemical detection. Site-controllable immobilization of protein was realized in a 30 mm long microfluidic chip channel by the strategy to create a approximately 1.7 mm concentrated FITC-BSA band, which leads to great improvement of the elute peak shape, accomplished with remarkably increased sensitivity, approximately 20 times higher than that without IEF-GEL treatment to GOx reactors. The kinetic response of GOx after IEF-GEL treatment was also investigated. The proposed system holds the advantages of IEF and low-temperature sol-gel technologies, i.e. concentrating the protein to be focused and retaining the biological activity for the gel-embedded protein, thus realizes site-specific immobilization of low-concentration protein at nL volume level.