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Sample records for binding site-specific evolutionarymodel

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-10-28

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

  2. Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in vitro

    Science.gov (United States)

    Cooney, Michael; Czernuszewicz, Graznya; Postel, Edith H.; Flint, S. Jane; Hogan, Michael E.

    1988-07-01

    A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

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

    Science.gov (United States)

    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.

  4. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  5. CLONING AND CHARACTERIZATION OF A NUCLEAR, SITE-SPECIFIC SSDNA BINDING-PROTEIN

    NARCIS (Netherlands)

    SMIDT, MP; RUSSCHEN, B; SNIPPE, L; WIJNHOLDS, J; AB, G

    1995-01-01

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of

  6. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  7. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    Science.gov (United States)

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  8. Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

    International Nuclear Information System (INIS)

    The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity

  9. Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Brandon L Jutras

    Full Text Available A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.

  10. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

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

    2015-09-01

    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.

  11. Detection of site-specific binding and co-binding of ligands to macromolecules using sup 19 F NMR

    Energy Technology Data Exchange (ETDEWEB)

    Jenkins, B.G. (Massachusetts General Hospital and Harvard Medical School, Charlestown (USA))

    1991-01-01

    Study of ligand-macromolecular interactions by {sup 19}F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of {sup 19}F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple {sup 19}F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of {sup 19}F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed.

  12. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  13. Molecular recognition: monomer of the yeast transcriptional activator GCN4 recognizes its dimer DNA binding target sites specifically

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It is widely believed that dimerization is a requirement for the yeast transcriptional activator GCN4 to recognize its specific DNA target sites. We used the basic region (226-252) of the yeast transcriptional activator GCN4, as both a monomeric peptide and a disulfide-linked dimer to investigate the interaction of the peptides with the DNA target sites AP-1 and CRE. CD and ITC experiments indicate that although the monomeric peptide GCN4-M has a weaker affinity with the DNA relative to the disulfide-linked dimer peptide GCN4-D, it recognizes AP-1 and CRE target sites specifically.

  14. Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.

    OpenAIRE

    Yu, A.; Haggård-Ljungquist, E

    1993-01-01

    Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both t...

  15. Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry.

    Science.gov (United States)

    Ren, Xiao-Min; Cao, Lin-Ying; Zhang, Jing; Qin, Wei-Ping; Yang, Yu; Wan, Bin; Guo, Liang-Hong

    2016-04-01

    Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.

  16. Site-Specific Innovation

    DEFF Research Database (Denmark)

    Reeh, Henrik; Hemmersam, Peter

    2015-01-01

    Currently, cities across the Northern European region are actively redeveloping their former industrial harbours. Indeed, harbours areas are essential in the long-term transition from industrial to information and experience societies; harbours are becoming sites for new businesses and residences...... question is how innovation may contribute to urban life and site-specific qualities....

  17. Identification of a novel site specific endonuclease produced by Mycoplasma fermentans: discovery while characterizing DNA binding proteins in T lymphocyte cell lines.

    OpenAIRE

    Halden, N F; Wolf, J B; Leonard, W J

    1989-01-01

    We have discovered a new restriction endonuclease, MfeI, in nuclear extracts from T cells contaminated with Mycoplasma fermentans. This endonuclease was identified while studying proteins binding to the interleukin-2 receptor alpha chain gene promoter. MfeI cuts at the recognition sequence C'AATTG generating EcoRI compatible cohesive ends. Potential applications are discussed.

  18. Structural transformation induced by locked nucleic acid or 2′–O-methyl nucleic acid site-specific modifications on thrombin binding aptamer

    OpenAIRE

    Liu, Bo; Li, Da

    2014-01-01

    Background Locked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades. However, how they affect DNA quadruplex structures remains largely unknown. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect G-quadruplex structure formation using a thrombin binding aptamer (TBA), the most studied extracorporal G-quadruplex-forming DNA sequence, whi...

  19. Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts

    OpenAIRE

    Pfalz, Jeannette; Bayraktar, Omer Ali; Prikryl, Jana; Barkan, Alice

    2009-01-01

    Chloroplast mRNA populations are characterized by overlapping transcripts derived by processing from polycistronic precursors. The mechanisms and functional significance of these processing events are poorly understood. We describe a pentatricopeptide repeat (PPR) protein, PPR10, whose binding defines mRNA segments derived from two transcription units in maize chloroplasts. PPR10 interacts in vivo and in vitro with two intergenic RNA regions of similar sequence. The processed 5′ and 3′ RNA te...

  20. Thermodynamic basis of site-specific cooperativity.

    Science.gov (United States)

    Di Cera, E

    1994-08-01

    Cooperative phenomena in biological macromolecules arise from the interaction of many distinct subsystems, such as structural domains or binding sites. Cooperative properties of the system as a whole, like protein folding or allosteric transitions, are subject to the restrictions imposed by thermodynamic stability. These restrictions, however, do not apply in the case of individual subsystems open to interactions with the rest of the macromolecule. The site-specific properties of such subsystems can be understood in general thermodynamic terms from those of a multicomponent system under particular conditions. The analogy provides a thermodynamic basis for site-specific cooperativity. PMID:8075382

  1. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone.

    Science.gov (United States)

    Shabanpoor, Fazel; Bathgate, Ross A D; Belgi, Alessia; Chan, Linda J; Nair, Vinojini B; Wade, John D; Hossain, Mohammed Akhter

    2012-04-01

    Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide. PMID:22425984

  2. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

    International Nuclear Information System (INIS)

    Highlights: ► A mono-Eu-DTPA conjugated peptide ligand, Eu-DTPA-(A)-H2, has been developed. ► The choice of a site for incorporation of a chelator is critical. ► The labeled peptide retains full activity at the RXFP1 receptor. ► It is markedly cheaper to produce and easier to use than radioactive probes. -- Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure–activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand–receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of

  3. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

    Energy Technology Data Exchange (ETDEWEB)

    Shabanpoor, Fazel [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia); Bathgate, Ross A.D.; Belgi, Alessia [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC 3010 (Australia); Chan, Linda J.; Nair, Vinojini B.; Wade, John D. [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia); Hossain, Mohammed Akhter, E-mail: akhter.hossain@florey.edu.au [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer A mono-Eu-DTPA conjugated peptide ligand, Eu-DTPA-(A)-H2, has been developed. Black-Right-Pointing-Pointer The choice of a site for incorporation of a chelator is critical. Black-Right-Pointing-Pointer The labeled peptide retains full activity at the RXFP1 receptor. Black-Right-Pointing-Pointer It is markedly cheaper to produce and easier to use than radioactive probes. -- Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to

  4. Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

    Science.gov (United States)

    Lipps, Georg; Stegert, Mario; Krauss, Gerhard

    2001-01-01

    There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C. PMID:11160922

  5. Site specific agreement : Lake Mason Wetland

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The site-specific agreement describes purpose and scope of the partnership between Ducks Unlimited, Inc. and U.S. Fish and Wildlife Service to manage wetlands for...

  6. Characterising site-specific water availability

    OpenAIRE

    Duda, Regine

    2005-01-01

    The leaf water potential and the soil matric potential as well as the silicon accumulation in leaves were studied as characteristics of site-specific water availability. Site-specific water availability could be described in a semi-quantitative way when the soil matric potential was correlated to the midday leaf water potential. Crop transpiration, hence water availability, could be roughly estimated from the changes in the Si-content of leaves. Therefore this parameter may rather be able to ...

  7. The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein

    Science.gov (United States)

    Lipps, Georg; Ibanez, Pablo; Stroessenreuther, Thomas; Hekimian, Katya; Krauss, Gerhard

    2001-01-01

    The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by ∼60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins. PMID:11812827

  8. Site-specific gene modification by PNAs conjugated to psoralen.

    Science.gov (United States)

    Kim, Ki-Hyun; Nielsen, Peter E; Glazer, Peter M

    2006-01-10

    DNA-binding molecules, including triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs), can be utilized to introduce site-specific mutations or to promote recombination at selected genomic sites. To further evaluate the utility of PNAs for site-specific gene modification, we tested dimeric bis-PNAs conjugated to psoralen. These PNAs are designed to form a triplex-invasion complex within the supF reporter gene in an episomal shuttle vector and to direct site-specific photoadduct formation by the conjugated psoralen. The psoralen-bis-PNA conjugate was found to direct photoadduct formation to the intended 5'-TpA base step next to the PNA-binding site, and the photoadduct formation efficiency displayed both concentration and UVA irradiation dependence. The effect of PNA-targeted photoadducts in a mammalian system was tested by SV40-based shuttle vector assay. After in vitro binding, we found that photoadducts directed by PNAs conjugated to psoralen-induced mutations at frequencies in the range of 0.46%, 6.5-fold above the background. In a protocol for intracellular gene targeting in the episomal shuttle vector, the psoralen-PNA-induced mutation frequency was 0.13%, 3.5-fold higher than the background. Most of the induced mutations were deletions and single-base-pair substitutions at or adjacent to the targeted PNA-binding and photoadduct-formation sites. When the results are taken together, they demonstrate the ability of bis-PNAs conjugated with psoralen to mediate site-specific gene modification, and they further support the development of PNAs as tools for gene-targeting applications. PMID:16388608

  9. Site-specificity in small architectural interventions

    DEFF Research Database (Denmark)

    Marie Bruun Jespersen, Line

    2016-01-01

    This paper is investigating the concepts of site-specificity, place and experience as they have been interpreted in a number of projects with at strong emphasis on the characteristics of the site, in the form of small architectural or artistic interventions. The idea of site-specificity is re......-emerging in contemporary architectural discourse in a series of projects, in e.g. the Norwegian project ”National Tourist Roads”. The small architectural interventions consequently work with enhancing or framing the qualities of site and aim at being a mediating element between man and nature. In this paper two main...... specificity in the small architectural interventions. The paper concludes that the small architectural interventions operate with a narrow concept of site, and that the large variety of artistic strategies and types of site-specificity found in contemporary art could serve as inspiration to open up...

  10. DOE site-specific threat assessment

    International Nuclear Information System (INIS)

    A facility manager faced with the challenges of protecting a nuclear facility against potential threats must consider the likelihood and consequences of such threats, know the capabilities of the facility safeguards and security systems, and make informed decisions about the cost-effectivness of safeguards and security upgrades. To help meet these challenges, the San Francisco Operations Office of the Department of Energy, in conjunction with the Lawrence Livermore Laboratory, has developed a site-specific threat assessment approach and a quantitative model to improve the quality and consistency of site-specific threat assessment and resultant security upgrade decisions at sensitive Department of Energy facilities. 5 figs

  11. Prospects for site specific weed management

    DEFF Research Database (Denmark)

    Christensen, Svend; Rasmussen, Jesper; Pedersen, Søren Marcus;

    2014-01-01

    Research on Site Specific Weed Management (SSWM) started in the late 80's. Since that moment, considerable research has been conducted on different aspects of SSWM, from fundamental studies on the spatial ecology of weeds to the applied development and testing of new technologies for weed detection...

  12. Nanoparticles for Site Specific Genome Editing

    Science.gov (United States)

    McNeer, Nicole Ali

    Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

  13. Site-specific ground response analysis

    OpenAIRE

    GovindaRaju, L; RAMANA, GV; HanumanthaRao, C; Sitharam, TG

    2004-01-01

    The local soil conditions have a profound influence on ground response during earthquakes. The recent destructive earthquakes have again demonstrated that the topography, nature of the bedrock and nature and geometry of the depositional soils are the primary factors thatinfluence local modifications to the underlying motion. We highlight the engineering importance of site-specific ground response analysis and difficulties faced in conducting a complete ground response analysis. Steps to be fo...

  14. Site-Specific PEGylation of Therapeutic Proteins

    Directory of Open Access Journals (Sweden)

    Jonathan K. Dozier

    2015-10-01

    Full Text Available The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

  15. Savannah River Site's Site Specific Plan

    International Nuclear Information System (INIS)

    This Site Specific Plan (SSP) has been prepared by the Savannah River Site (SRS) in order to show the Environmental Restoration and Waste Management activities that were identified during the preparation of the Department of Energy-Headquarters (DOE-HQ) Environmental Restoration and Waste Management Five-Year Plan (FYP) for FY 1992--1996. The SSP has been prepared in accordance with guidance received from DOE-HQ. DOE-SR is accountable to DOE-HQ for the implementation of this plan. The purpose of the SSP is to develop a baseline for policy, budget, and schedules for the DOE Environmental Restoration and Waste Management activities. The plan explains accomplishments since the Fiscal Year (FY) 1990 plan, demonstrates how present and future activities are prioritized, identifies currently funded activities and activities that are planned to be funded in the upcoming fiscal year, and describes future activities that SRS is considering

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

    Science.gov (United States)

    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.

  17. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    Science.gov (United States)

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. PMID:27315553

  18. Appreciating Site-Specific Qualities in Urban Harbours

    DEFF Research Database (Denmark)

    Reeh, Henrik

    2015-01-01

    When “site-specificity” becomes a central value in city and harbor transformation, it soon proves necessary to address the ways in which scholars and professionals actually determine site-specific qualities in urban fabrics and social life. This paper delves into the above questions by means of o...... of site-specificity, even in the traditional harbor settings. Considered with conceptual care, such situations may teach us what it means to “appreciate site-specific qualities”....

  19. 77 FR 22772 - Environmental Management Site-Specific Advisory Board

    Science.gov (United States)

    2012-04-17

    ... Environmental Management Site-Specific Advisory Board AGENCY: Office of Environmental Management, Department of... Administration, notice is hereby given that the Environmental Management Site-Specific Advisory Board (EM SSAB... recommendations to the Assistant Secretary for Environmental Management (EM) concerning issues affecting the......

  20. Innovation and Diffusion of Site-specific Crop Management

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus; Pedersen, Jørgen Lindgaard

    2006-01-01

    Site-specific crop management or precision farming is a highly complex managementsystem for site-specific input application of lime, fertilizers and pesticides in arable farming. The Global Positioning System (GPS)is the backbone of the system. To conduct precision farming several technical systems...

  1. Site-Specific Chemical Labeling of Long RNA Molecules

    DEFF Research Database (Denmark)

    Jahn, Kasper; Olsen, Eva Maria; Nielsen, Morten Muhlig;

    2011-01-01

    is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor......Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method......-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range...

  2. Site-Specific Recombination Strategies for Engineering Actinomycete Genomes

    OpenAIRE

    Herrmann, Simone; Siegl, Theresa; Luzhetska, Marta; Petzke, Lutz; Jilg, Caroline; Welle, Elisabeth; Erb, Annette; Leadlay, Peter F; Bechthold, Andreas; Luzhetskyy, Andriy

    2012-01-01

    The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been ut...

  3. Protein assemblies by site-specific avidin-biotin interactions.

    Science.gov (United States)

    Mori, Yutaro; Minamihata, Kosuke; Abe, Hiroki; Goto, Masahiro; Kamiya, Noriho

    2011-08-21

    Exploiting self-assembly systems with biological building blocks is of significant interest in the fabrication of advanced biomaterials. We assessed the potential use of site-specific ligand labeling of protein building blocks in designing functional protein self-assemblies by combining site-specifically biotinylated bacterial alkaline phosphatase (as a bidentate or tetradentate ligand unit) and streptavidin (as a tetrameric receptor). PMID:21731938

  4. Site-specific recombinases as tools for heterologous gene integration.

    Science.gov (United States)

    Hirano, Nobutaka; Muroi, Tetsurou; Takahashi, Hideo; Haruki, Mitsuru

    2011-10-01

    Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes. PMID:21822899

  5. Innovation and diffusion of site-specific crop management

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus; Pedersen, Jørgen Lindgaard

    2004-01-01

    Site-specific crop management or precision farming (PF) is a highly complex management system for site-specific input application of lime, fertilizers and pesticides in arable farming. The Global Positioning System (GPS) is the backbone of the system. To conduct PF several technical systems...... and data interpretations are needed. FP is aiming to be used by large farms because of scale advantages. In reality adoption and benefits harvested are limited. We have analysed information from 28 experts and stakeholders and focus group meetings with 35 participants and a multinational farm survey...

  6. INVESTMENT IN SITE SPECIFIC CROP MANAGEMENT UNDER UNCERTAINTY

    OpenAIRE

    Isik, Murat; Khanna, Madhu; Winter-Nelson, Alex

    1999-01-01

    This paper examines the impact of price uncertainty on farmers' adoption decision in site specific crop management using an option value model. It shows that price uncertainty could lead farmers to delay the investment 3 to 25 years as opposed to the net present value rule. Immediate investment is only worthwhile on high soil fertility and soil quality fields with high variability.

  7. Discovering Site-Specific Qualities in Venice and Marseille

    DEFF Research Database (Denmark)

    Reeh, Henrik

    2015-01-01

    When “site-specificity” becomes a central value in city and harbor transfor-mation, it soon proves necessary to address the ways in which scholars and professionals actually come to determine site-specific qualities in urban fab-rics and social life. How are certain traits and habits discovered...

  8. Multispectral Imaging Technology for Site-Specific Crop Fertilization

    Science.gov (United States)

    Agricultural fields are variable and require site-specific crop management. Nitrogen (N) is an essential nutrient required for plant growth and is a major component of the chlorophyll molecule enhancing photosynthesis. However, excessive N fertilizer leaches into groundwater and creates serious envi...

  9. Site-specific cleavage of genomic DNA mediated by triple helix formation

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, S.A.

    1992-01-01

    Physical isolation of large segments of chromosomal DNA is a major goal of human genetics. This would be greatly assisted by a generalizable technique for the cleavage of chromosomal DNA at a single site. Pyrimidine oligonucleotide directed triple helix formation is a generalizable motif for the site specific recognition of duplex DNA. This thesis describes the application of oligonucleotide directed triple helix formation to bind unique target sites in bacteriophage [lambda], yeast, and human genomic DNA. Cleavage at the binding sites are achieved by affinity cleaving with EDTA[center dot]Fe(II) derivatized oligonucleotides, alkylation with bromoacetyl derivatized oligonucleotides, and by site specific triple helix mediated methylase inhibition followed by digestion with the cognate endonuclease. Cleavage of genomic substrates with progressively greater complexity is described. Bacteriophage [lambda] genomic DNA (48.5 kilobase pairs) was targeted at a single endogenous homopurine site within the origin of replication. This substrate was also used to demonstrate cooperative binding of heterologous oligonucleotides to duplex DNA at contiguous binding sites. An engineered target site on yeast chromosome III (340 kilobase pairs) was cut quantitatively at a single site within total yeast genomic DNA (14 megabase pairs) by both chemical and enzymatic techniques. Techniques for the identification of endogenous triple helix target sites within unsequenced genetic markers were developed and successfully used to characterize a target site on human chromosome 4, proximal to the Huntington disease gene. As a test for the site specific cleavage of gigabase DNA, this site near the end of human chromosome 4 was cleaved by triple helix mediated enzymatic cleavage. This generated a specific 3.6 Mb fragment in greater than 80% yield that contained the entire candidate region for the Huntington mutation.

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

    Science.gov (United States)

    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. Drainage filter technologies to mitigate site-specific phosphorus losses

    DEFF Research Database (Denmark)

    Kjærgaard, Charlotte; Heckrath, Goswin Johann; Iversen, Bo Vangsø;

    2014-01-01

    high risks areas of P loss and applying site-specific measures therefore seems a more cost-efficient approach. The Danish Commission for Nature and Agriculture has now called for a shift of paradigm towards targeted mitigation and development of new, cost-efficient technologies to mitigate site......-specific nutrient losses in drainage. The “SUPREME-TECH” project (2010-2015), funded by the Danish Strategic Research Council, aims at providing the scientific basis for developing cost-effective drainage filter technologies to retain P in agricultural drainage waters. The project studies different approaches......-scale surface-flow constructed wetland. In the former, various natural and industrial P filter substrates have been tested for their ability to reduce inlet P concentrations to below environmental threshold values (

  12. SITE-94. Site specific base data for the performance assessment

    Energy Technology Data Exchange (ETDEWEB)

    Geier, J. [ed.] [Clearwater Hardrock Consulting, Monmouth, OR (United States); Tiren, S. [Geosigma AB, Uppsala (Sweden); Dverstorp, B. [Swedish Nuclear Power Inspectorate, Stockholm (Sweden); Glynn, P. [U.S. Geological Survey, Reston, VA (United States)

    1996-06-01

    This report documents the site specific base data that were available, and the utilization of these data within SITE-94. A brief summary is given of SKB`s preliminary site investigations for the Aespoe Hard Rock Laboratory (HRL), which were the main source of site-specific data for SITE-94, and an overview is given of the field methods and instrumentation for the preliminary investigations. A compilation is given of comments concerning the availability and quality of the data for Aespoe, and specific recommendations are given for future site investigations. It was found that the HRL pre-investigations produced a large quantity of data which were, for the most part, of sufficient quality to be valuable for a performance assessment. However, some problems were encountered regarding documentation, procedural consistency, positional information, and storage of the data from the measurements. 77 refs, 4 tabs.

  13. Site-specific design optimization of wind turbines

    DEFF Research Database (Denmark)

    Fuglsang, P.; Bak, C.; Schepers, J.G.;

    2002-01-01

    This article reports results from a European project, where site characteristics were incorporated into the design process of wind turbines, to enable site-specific design. Two wind turbines of different concept were investigated at six different sites comprising normal flat terrain, offshore...... of a new wind turbine. Both annual energy yield and design-determining loads depended on site characteristics, and this represented a potential for site-specific design. The maximum variation in annual energy yield was 37% and the maximum variation in blade root fatigue loads was 62%. Optimized site...... advantage of high-wind-speed sites. It was not possible to design a single wind turbine for all wind climates investigated, since the differences in the design loads were too large. Multiple-site wind turbines should be designed for generic wind conditions, which cover wind parameters encountered at flat...

  14. DNA Cloning Using In Vitro Site-Specific Recombination

    OpenAIRE

    Hartley, James L.; Temple, Gary F.; Brasch, Michael A.

    2000-01-01

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic...

  15. Site-specific features influence sediment stability of intertidal flats

    OpenAIRE

    Defew, E. C.; T. J. Tolhurst; D. M. Paterson

    2002-01-01

    The factors that influence the sediment stability and the transport of estuarine mudflats are not yet fully understood but knowledge of them is essential in coastal engineering applications and pollution ecology studies. The suggestion that variation in predictive models of sediment stability might be due to site-specific characteristics is investigated using data from four estuarine mudflats (Eden Estuary, Scotland, the Biezelingsche Ham, Zandkreek, and Molenplaat mudflats in The Netherlands...

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

    Energy Technology Data Exchange (ETDEWEB)

    Zope, Rajendra R., E-mail: rzope@utep.edu; Baruah, Tunna [Department of Physics, The University of Texas at El Paso, El Paso, Texas 79958 (United States); Computational Science Program, The University of Texas at El Paso, El Paso, Texas 79958 (United States); Bhusal, Shusil; Basurto, Luis [Department of Physics, The University of Texas at El Paso, El Paso, Texas 79958 (United States); Jackson, Koblar [Physics Department and Science of Advanced Materials Ph.D. Program, Central Michigan University, Mt. Pleasant, Michigan 48859 (United States)

    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 isolated 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.

  17. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Sass, Ehud; Suski, Catherine;

    2014-01-01

    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications......., to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it...

  18. Site-Specific ecological risk assessment. Case-study 2

    DEFF Research Database (Denmark)

    Jensen, John

    The decision supporting and integrating assessment tool, TRIAD, is used site-specific on PAH- and heavy metal contaminated sites in Denmark. The various aspects of the TRIAD approach are used on a set of chemistry-, ecotoxicology- and ecology related data collected among others in the EU project...... “Development of a decision support system for sustainable management of contaminated land by linking bioavailability, ecological risk and ground water pollution of organic pollutants”or in short “LIBERATION”. The presentation includes examples on how to scale and integrate the results from various scientific...

  19. Determination of site-specific glycan heterogeneity on glycoproteins

    DEFF Research Database (Denmark)

    Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich;

    2012-01-01

    site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco......The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their...

  20. Site specific management in an olive tree plantation

    DEFF Research Database (Denmark)

    Fountas, S.; Aggelopoulou, K.; Bouloulis, C.;

    2011-01-01

    shoots and letting the olives fall onto a plastic net covering the ground. Sacks of approximately 58 kg capacity were filled with olives from as many adjacent trees as were needed to fill a sack. The location of the sacks, or group of closely placed sacks, was identified using a commercial GPS (5 m....... Maps were created as a basis for site-specific management of P, K and lime, and these were applied 15 days after harvest in the winter of 2008. The results indicated considerable spatial variation in yield and soil properties. The soil organic matter content was about 22% greater and the penetration...

  1. Site Specific Probable Maximum Precipitation Estimates and Professional Judgement

    Science.gov (United States)

    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

  2. Chemical tags for site-specific fluorescent labeling of biomolecules.

    Science.gov (United States)

    Freidel, Christoph; Kaloyanova, Stefka; Peneva, Kalina

    2016-06-01

    This review focuses on the various approaches to covalently attach a chromophore to a biomolecule of interest in site-specific manner. Novel methods like inverse electron-demand Diels-Alder reaction, Pictet-Spengler ligation and enzyme tags like SNAP and Halo-tags are critically discussed and compared to established techniques like copper-free click reaction and native chemical ligation. Selected examples in which the tags have been exploited for in vitro or in vivo imaging are reviewed and evaluated. PMID:26969255

  3. Site-specific features influence sediment stability of intertidal flats

    Science.gov (United States)

    Defew, Emma C.; Tolhurst, Trevor J.; Paterson, David M.

    The factors that influence the sediment stability and the transport of estuarine mudflats are not yet fully understood but knowledge of them is essential in coastal engineering applications and pollution ecology studies. The suggestion that variation in predictive models of sediment stability might be due to site-specific characteristics is investigated using data from four estuarine mudflats (Eden Estuary, Scotland, the Biezelingsche Ham, Zandkreek, and Molenplaat mudflats in The Netherlands). These estuaries differ in their environmental conditions, macrofaunal species composition and local features (e.g. Enteromorpha mats, migratory biofilms). Stable and unstable sediments were compared, and mean chlorophyll-a concentrations and granulometry of the sediments were significantly different between the two groups. Step-wise multiple linear regressions were applied to the sediment stability data of all sites to establish the influences on erosion threshold of microphytobenthic biomass, water content, granulometry, organic carbon content and the abundance of dominant macrofaunal species. The stability of each site was influenced by different factors. Sediment stability of the Eden Estuary was affected by the Enteromorpha bloom; Biezelingsche Ham was influenced by the highly migratory nature of the diatom biofilms and the abundance of Corophium volutator; the polychaete worm Arenicola marina had a net negative effect on sediment stability of the Zandkreek; and the Molenplaat was influenced by microphytobenthic biomass. This research highlights the need for site-specific calibration of models and suggests that a universal proxy parameter for sediment stability is unlikely to be obtained.

  4. Temporally-controlled site-specific recombination in zebrafish.

    Directory of Open Access Journals (Sweden)

    Stefan Hans

    Full Text Available 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 CreER(T2. 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.

  5. Site-specific Probabilistic Seismic Hazard Map of Himachal Pradesh, India. Part I. Site-specific Ground Motion Relations

    Directory of Open Access Journals (Sweden)

    Muthuganeisan Prabhu

    2016-04-01

    Full Text Available This article presents four regional site-specific ground motion relations developed for the state of Himachal Pradesh in northwest Himalaya, situated in a seismically active region. These relations are developed from synthetic free surface ground motion databases obtained from a calibrated stochastic seismological model considering the characteristic properties of this specific region. The adopted methodology incorporates the site effects characterised through active MASW tests conducted in 22 important cities. The estimated ground motion levels from the developed relations are found to be in reasonable agreement with the recorded data.

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

    Science.gov (United States)

    Merkert, Sylvia; Martin, Ulrich

    2016-01-01

    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. PMID:27347935

  7. Site-specific recombinases: molecular machines for the Genetic Revolution.

    Science.gov (United States)

    Olorunniji, Femi J; Rosser, Susan J; Stark, W Marshall

    2016-03-15

    The fields of molecular genetics, biotechnology and synthetic biology are demanding ever more sophisticated molecular tools for programmed precise modification of cell genomic DNA and other DNA sequences. This review presents the current state of knowledge and development of one important group of DNA-modifying enzymes, the site-specific recombinases (SSRs). SSRs are Nature's 'molecular machines' for cut-and-paste editing of DNA molecules by inserting, deleting or inverting precisely defined DNA segments. We survey the SSRs that have been put to use, and the types of applications for which they are suitable. We also discuss problems associated with uses of SSRs, how these problems can be minimized, and how recombinases are being re-engineered for improved performance and novel applications. PMID:26965385

  8. Site-specific features influence sediment stability of intertidal flats

    Directory of Open Access Journals (Sweden)

    E. C. Defew

    2002-01-01

    Full Text Available The factors that influence the sediment stability and the transport of estuarine mudflats are not yet fully understood but knowledge of them is essential in coastal engineering applications and pollution ecology studies. The suggestion that variation in predictive models of sediment stability might be due to site-specific characteristics is investigated using data from four estuarine mudflats (Eden Estuary, Scotland, the Biezelingsche Ham, Zandkreek, and Molenplaat mudflats in The Netherlands. These estuaries differ in their environmental conditions, macrofaunal species composition and local features (e.g. Enteromorpha mats, migratory biofilms. Stable and unstable sediments were compared, and mean chlorophyll-a concentrations and granulometry of the sediments were significantly different between the two groups. Step-wise multiple linear regressions were applied to the sediment stability data of all sites to establish the influences on erosion threshold of microphytobenthic biomass, water content, granulometry, organic carbon content and the abundance of dominant macrofaunal species. The stability of each site was influenced by different factors. Sediment stability of the Eden Estuary was affected by the Enteromorpha bloom; Biezelingsche Ham was influenced by the highly migratory nature of the diatom biofilms and the abundance of Corophium volutator; the polychaete worm Arenicola marina had a net negative effect on sediment stability of the Zandkreek; and the Molenplaat was influenced by microphytobenthic biomass. This research highlights the need for site-specific calibration of models and suggests that a universal proxy parameter for sediment stability is unlikely to be obtained. Keywords: sediment stability; erosion threshold; cohesive strength meter; microphytobenthos; Enteromorpha spp.

  9. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination.

    Science.gov (United States)

    Thompson, J F; de Vargas, L M; Skinner, S E; Landy, A

    1987-06-01

    The highly directional site-specific recombination of bacteriophage lambda is tightly regulated by the binding of three different proteins to a complex array of sites. The manner in which these reactions are both stimulated and inhibited by co-operative binding of proteins to specific sites on the P arm of attP and AttR has been elucidated by correlation of nuclease protection with recombination studies of both wild-type and mutant DNAs. In addition to co-operative forces, there is a specific competitive interaction that allows the protein-DNA complex to serve as a "biological switch". This switch does not depend upon the simple occlusion of DNA binding sites by neighboring proteins; but, rather, the outcome of this competition is dependent on long-range interactions that vary between the higher-order structures of attP and attR. These higher-order structures are dependent on cooperative interactions involving three proteins binding to five or more sites. PMID:2958633

  10. Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase. Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme.

    Science.gov (United States)

    Adolph, H W; Maurer, P; Schneider-Bernlöhr, H; Sartorius, C; Zeppezauer, M

    1991-11-01

    different steps of the kinetic pathway have been investigated. The free energy of activation is higher for alcohol oxidation and lower for ketone reduction when catalyzed by Co(II)LADH in comparison to Zn(II)LADH. However, the free energies of binding are affected by metal substitution in such a way that the enantioselectivity of ketone reduction is not significantly changed by the substitution of Co(II) for Zn(II). 6. Evaluation of the data shows that substrate specificity and stereoselectivity result from combination of the free energies of binding and activation, with differences in binding energies as the dominating factors. In this regard, the interactions of substrate molecules with the protein moiety are dominant over the interactions with the catalytic metal ion.

  11. Site-specific basal body duplication in Chlamydomonas.

    Science.gov (United States)

    O'Toole, Eileen T; Dutcher, Susan K

    2014-02-01

    Correct centriole/basal body positioning is required for numerous biological processes, yet how the cell establishes this positioning is poorly understood. Analysis of centriolar/basal body duplication provides a key to understanding basal body positioning and function. Chlamydomonas basal bodies contain structural features that enable specific triplet microtubules to be specified. Electron tomography of cultures enriched in mitotic cells allowed us to follow basal body duplication and identify a specific triplet at which duplication occurs. Probasal bodies elongate in prophase, assemble transitional fibers (TF) and are segregated with a mature basal body near the poles of the mitotic spindle. A ring of nine-singlet microtubules is initiated at metaphase, orthogonal to triplet eight. At telophase/cytokinesis, triplet microtubule blades assemble first at the distal end, rather than at the proximal cartwheel. The cartwheel undergoes significant changes in length during duplication, which provides further support for its scaffolding role. The uni1-1 mutant contains short basal bodies with reduced or absent TF and defective transition zones, suggesting that the UNI1 gene product is important for coordinated probasal body elongation and maturation. We suggest that this site-specific basal body duplication ensures the correct positioning of the basal body to generate landmarks for intracellular patterning in the next generation. PMID:24166861

  12. The Application of TAPM for Site Specific Wind Energy Forecasting

    Directory of Open Access Journals (Sweden)

    Merlinde Kay

    2016-02-01

    Full Text Available The energy industry uses weather forecasts for determining future electricity demand variations due to the impact of weather, e.g., temperature and precipitation. However, as a greater component of electricity generation comes from intermittent renewable sources such as wind and solar, weather forecasting techniques need to now also focus on predicting renewable energy supply, which means adapting our prediction models to these site specific resources. This work assesses the performance of The Air Pollution Model (TAPM, and demonstrates that significant improvements can be made to only wind speed forecasts from a mesoscale Numerical Weather Prediction (NWP model. For this study, a wind farm site situated in North-west Tasmania, Australia was investigated. I present an analysis of the accuracy of hourly NWP and bias corrected wind speed forecasts over 12 months spanning 2005. This extensive time frame allows an in-depth analysis of various wind speed regimes of importance for wind-farm operation, as well as extreme weather risk scenarios. A further correction is made to the basic bias correction to improve the forecast accuracy further, that makes use of real-time wind-turbine data and a smoothing function to correct for timing-related issues. With full correction applied, a reduction in the error in the magnitude of the wind speed by as much as 50% for “hour ahead” forecasts specific to the wind-farm site has been obtained.

  13. Site-specific criteria for the completion of landfill aftercare.

    Science.gov (United States)

    Laner, David; Fellner, Johann; Brunner, Paul H

    2012-09-01

    Municipal solid waste (MSW) landfills need to be managed after closure to assure long-term environmental compatibility. Aftercare can be completed when the authorities consider the landfill not likely to pose a threat to humans and the environment. In this work, a methodology for deriving site-specific aftercare completion criteria is presented and its application is illustrated via a case study. The evaluation method combines models addressing waste emission behavior, long-term barrier performance, and pollutant migration to assess the potential impact of landfill emissions on the environment. Based on the definition of acceptable impact levels at certain points of compliance, scenario- and pollutant-specific aftercare completion criteria are derived. The methodology was applied to a closed MSW landfill in Austria and potential aftercare durations were determined. While landfill gas emissions may become environmentally tolerable within decades at the site, leachate-related aftercare measures were expected to be necessary for centuries (primarily as a result of ammonium). Although the evaluation comes with large uncertainties, it allows for linking aftercare intensity and duration with respect to an environmentally compatible state of the landfill in the absence of aftercare. However, further case studies including regulatory review and acceptance are needed to use the methodology in a decision support tool on aftercare completion.

  14. High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation.

    Science.gov (United States)

    Li, Xiaojuan; Xu, Wei; Wang, Yi; Zhao, Jia; Liu, Yan-Hui; Richardson, Daisy; Li, Huijuan; Shameem, Mohammed; Yang, Xiaoyu

    2016-08-19

    Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC-MS methods, which normally require a long (up to 24h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within antibody oxidation in real time stability studies. PMID:27432793

  15. Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

    Science.gov (United States)

    Qiu, Huawei; Boudanova, Ekaterina; Park, Anna; Bird, Julie J; Honey, Denise M; Zarazinski, Christine; Greene, Ben; Kingsbury, Jonathan S; Boucher, Susan; Pollock, Julie; McPherson, John M; Pan, Clark Q

    2013-03-20

    Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and β subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ~85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.

  16. 75 FR 56526 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2010-09-16

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION... 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...

  17. Site-Specific Seismic Site Response Model for the Waste Treatment Plant, Hanford, Washington

    International Nuclear Information System (INIS)

    This interim report documents the collection of site-specific geologic and geophysical data characterizing the Waste Treatment Plant site and the modeling of the site-specific structure response to earthquake ground motions

  18. Site-Specific Seismic Site Response Model for the Waste Treatment Plant, Hanford, Washington

    Energy Technology Data Exchange (ETDEWEB)

    Rohay, Alan C.; Reidel, Steve P.

    2005-02-24

    This interim report documents the collection of site-specific geologic and geophysical data characterizing the Waste Treatment Plant site and the modeling of the site-specific structure response to earthquake ground motions.

  19. Site-specific hydrogen diffusion rates during clinopyroxene dehydration

    Science.gov (United States)

    Ferriss, Elizabeth; Plank, Terry; Walker, David

    2016-06-01

    The rate of hydrogen diffusion in clinopyroxene is relevant to interpreting hydrogen ("water") concentrations in xenoliths, phenocrysts, and clinopyroxene-hosted melt inclusions to provide insight into the deep-earth water cycle and volcanic explosivity. Here, we determine bulk and site-specific hydrogen diffusivities in two diopsides and an augite by heating initially homogeneous water-bearing samples in a 1-atm CO/CO2 gas-mixing furnace at 800-1000 °C and oxygen fugacity at the quartz-fayalite-magnetite buffer and observing H-loss profiles. The O-H stretching range between wavenumbers 3000 and 4000 cm-1 in FTIR spectra is resolved into 4-6 peaks, each of which is assumed to represent a distinct defect site for the hydrogen, to determine peak-specific diffusivities using our previously published whole-block method. For the diopside from the Kunlun Mts. in China, Arrhenius relations are reported for peaks at 3645, 3617, 3540, 3443, and 3355 cm-1 based on measurements at 816, 904, and 1000 °C. Bulk and site-specific diffusivities are determined for the same set of peaks at 904 °C for the second diopside (Jaipur). The augite (PMR-53) was a triangular thin slab, and hydrogen diffusivities were determined for bulk hydrogen and peaks at 3620, 3550, 3460, and 3355 cm-1 in the thickness direction at 800 °C. Bulk hydrogen diffusivity in the Jaipur diopside is consistent with previous work, and hydrogen diffusivity in augite PMR-53 is slightly lower than the fast direction diffusivities measured || [100] and [001]* in Jaipur diopside. Both diopsides show 1-2 orders of magnitude differences in the peaks-specific diffusivities, with the fastest diffusivities at 3450 cm-1 and the slowest at 3645 cm-1. However, the hydrogen diffusivities in Jaipur diopside are 2-4 orders of magnitude higher than those in Kunlun diopside for bulk hydrogen and all peaks. Thus, peak-specific differences cannot by themselves adequately explain the 5 orders of magnitude range in hydrogen

  20. Multispectral videography for site-specific farm management

    Science.gov (United States)

    Anderson, Gerald L.; Yang, C.

    1996-11-01

    Researchers are expending considerable effort to develop the technology and methodology needed to identify and map within-field management zones for site-specific farming. Much of the research has focused on the use of either a high-density geographically referenced grid of soil samples or mechanical yield sensor measurements that record geographic positions and production levels. In either case, complex spatial models are generally used to extrapolate the various soil variables and production level information across the entire field. Both procedures produce a wealth of information, however, the analysis of soil samples tend to be quite expensive and the accuracy of mechanical yield measurements does vary. This study represents an ongoing effort designed to evaluate remote sensing as a tool for determining within field management zones. Color-infrared aerial photography and multispectral videography were used in concert to map and stratify two grain sorghum fields into regions or zones of homogeneous spectral response. A limited number of soil and plant samples were acquired to characterize the biotic and edaphic conditions within each zone. Results obtained during the first year of the study indicated that multispectral video can be used to develop within field management zones. Simple univariate analysis indicated that soil pH, Ca, and Fe were important variables affecting yield. Analysis of the yield data indicated that the economic returns from 17% of the first field and 20% of the second field were insufficient to recoup planting costs. Multispectral video also proved instrumental in modeling the spatial variability of yield. A significant negative correlation (r2 greater than 0.90) was obtained between the red spectral band and crop yields for both fields. Stratification, in this case using image data, reduces the number of samples required to characterize a field by reducing the variance associated within each stratum. Image data also provided a

  1. Oxygen as a site specific structural probe in neutron diffraction

    Energy Technology Data Exchange (ETDEWEB)

    Neuefeind, Joerg C [ORNL; Simonson, J Michael {Mike} [ORNL; Salmon, Phil [University of Bath; Zeidler, Anita [University of Bath; Fischer, Henry E [Institut Laue-Langevin (ILL); Rauch, Helmut [E141 Atominstitut der & #xD6; sterreichischen Universit& #xE4; ten,; Markland, Thomas [Columbia University; Lemmel, Hartmut [Technical University Vienna

    2011-01-01

    Oxygen is a ubiquitous element, playing an essential role in most scientific and technological disciplines, and is often incorporated within a structurally disordered material where examples include molten silicates in planetary science, glasses used for lasers and optical communication, and water in biological processes. Establishing the structure of a liquid or glassy oxide and thereby its relation to the functional properties of a material is not, however, a trivial task owing to the complexity associated with atomic disorder. Here we approach this challenge by measuring the bound coherent neutron scattering lengths of the oxygen isotopes with the sensitive technique of neutron interferometry. We find that there is a small but finite contrast of 0.204(6) fm between the scattering lengths of the isotope 18O and oxygen of natural isotopic abundance natO, contrary to tables of recommended values. This has enabled us to investigate the structure of both light and heavy water by exploiting, for the first time, the method of oxygen isotope substitution in neutron diffraction, thus circumventing many of the significant problems associated with more traditional methods in which hydrogen is substituted by deuterium. We find a difference of ~0.5% between the O-H and O-D intra-molecular bond distances which is much smaller than recent estimates based on diffraction data and is found to be in excellent agreement with path integral molecular dynamics simulations made with a flexible polarisable water model. Our results demonstrate the potential for using oxygen isotope substitution as a powerful and effective site specific probe in a plethora of materials, of pertinence as instrumentation at next generation neutron sources comes online

  2. Improving Site-Specific Radiological Performance Assessments - 13431

    Energy Technology Data Exchange (ETDEWEB)

    Tauxe, John; Black, Paul; Catlett, Kate; Lee, Robert; Perona, Ralph; Stockton, Tom; Sully, Mike [Neptune and Company, Inc., Los Alamos, New Mexico 87544 (United States)

    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, but 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

  3. Site-specific control of N7-metal coordination in DNA by a fluorescent purine derivative.

    Science.gov (United States)

    Dumas, Anaëlle; Luedtke, Nathan W

    2012-01-01

    A synthetic strategy that utilizes O6-protected 8-bromoguanosine gives broad access to C8-guanine derivatives with phenyl, pyridine, thiophene, and furan substituents. The resulting 8-substituted 2'-deoxyguanosines are push-pull fluorophores that can exhibit environmentally sensitive quantum yields (Φ=0.001-0.72) due to excited-state proton-transfer reactions with bulk solvent. Changes in nucleoside fluorescence were used to characterize metal-binding affinity and specificity of 8-substituted 2'-deoxyguanosines. One derivative, 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), exhibits selective binding of Cu(II), Ni(II), Cd(II), and Zn(II) through a bidentate effect provided by the N7 position of guanine and the 2-pyridyl nitrogen atom. Upon incorporation into DNA, 2-pyridine-modified guanine residues selectively bind to Cu(II) and Ni(II) with equilibrium dissociation constants (K(d)) that range from 25 to 850 nM; the affinities depend on the folded state of the oligonucleotide (duplex>G-quadruplex) as well as the identity of the metal ion (Cu>Ni≫Cd). These binding affinities are approximately 10 to 1 000 times higher than for unmodified metal binding sites in DNA, thereby providing site-specific control of metal localization in alternatively folded nucleic acids. Temperature-dependent circular-dichroism studies reveal metal-dependent stabilization of duplexes, but destabilization of G-quadruplex structures upon adding Cu(II) to 2PyG-modified oligonucleotides. These results demonstrate how the addition of a single pyridine group to the C8 position of guanine provides a powerful new tool for studying the effects of N7 metalation on the structure, stability, and electronic properties of nucleic acids.

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

    Science.gov (United States)

    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.

  5. Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

    Directory of Open Access Journals (Sweden)

    Paul Majkut

    Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

  6. Site-specific acetylation of ISWI by GCN5

    Directory of Open Access Journals (Sweden)

    Chioda Mariacristina

    2007-08-01

    Full Text Available Abstract Background The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. Results We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. Conclusion Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(KacxPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

  7. Site-specific IGFBP-1 hyper-phosphorylation in fetal growth restriction: clinical and functional relevance.

    Science.gov (United States)

    Abu Shehab, Majida; Khosravi, Javad; Han, Victor K M; Shilton, Brian H; Gupta, Madhulika B

    2010-04-01

    Phosphorylation enhances IGFBP-1 binding to IGF-I, thereby limiting the bioavailability of IGF-I that may be important in fetal growth. Our goal in this study was to determine whether changes in site-specific IGFBP-1 phosphorylation were unique to fetal growth restriction. To establish a link, we compared IGFBP-1 phosphorylation (sites and degree) in amniotic fluid from FGR (N = 10) and controls (N = 12). The concentration of serine phosphorylated IGFBP-1 showed a negative correlation with birth weight in FGR (P = 0.049). LC-MS/MS analysis revealed all four previously identified phosphorylation sites (Ser98, Ser101, Ser119, and Ser169) to be common to FGR and control groups. Relative phosphopeptide intensities (LC-MS) between FGR and controls demonstrated 4-fold higher intensity for Ser101 (P = 0.026), 7-fold for Ser98/Ser101 (P = 0.02), and 23-fold for Ser169 (P = 0.002) in the FGR group. Preliminary BIAcore data revealed 4-fold higher association and 1.7-fold lower dissociation constants for IGFBP-1/IGF-I in FGR. A structural model of IGFBP-1 bound to IGF-I indicates that all the phosphorylation sites are on relatively mobile regions of the IGFBP-1 sequence. Residues Ser98, Ser101, and Ser169 are close to structured regions that are involved in IGF-I binding and, therefore, could potentially make direct contact with IGF-I. On the other hand, residue Ser119 is in the middle of the unstructured linker that connects the N- and C-terminal domains of IGFBP-1. The model is consistent with the assumption that residues Ser98, Ser101, and Ser169 could directly interact with IGF-I, and therefore phosphorylation at these sites could change IGF-I interactions. We suggest that site-specific increase in IGFBP-1 phosphorylation limits IGF-I bioavailability, which directly contributes to the development of FGR. This study delineates the potential role of higher phosphorylation of IGFBP-1 in FGR and provides the basis to substantiate these findings with larger sample size. PMID

  8. In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures.

    Science.gov (United States)

    Shah, Rachit; Petersburg, Jacob; Gangar, Amit C; Fegan, Adrian; Wagner, Carston R; Kumarapperuma, Sidath C

    2016-07-01

    Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation, and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging, and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR(2)) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[(64)Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN's binding affinity toward U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of (64)Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished

  9. Site-specific tagging proteins with a rigid, small and stable transition metal chelator, 8-hydroxyquinoline, for paramagnetic NMR analysis

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yin; Huang, Feng [Nankai University, State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) (China); Huber, Thomas [Australian National University, Research School of Chemistry (Australia); Su, Xun-Cheng, E-mail: xunchengsu@nankai.edu.cn [Nankai University, State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) (China)

    2016-02-15

    Design of a paramagnetic metal binding motif in a protein is a valuable way for understanding the function, dynamics and interactions of a protein by paramagnetic NMR spectroscopy. Several strategies have been proposed to site-specifically tag proteins with paramagnetic lanthanide ions. Here we report a simple approach of engineering a transition metal binding motif via site-specific labelling of a protein with 2-vinyl-8-hydroxyquinoline (2V-8HQ). The protein-2V-8HQ adduct forms a stable complex with transition metal ions, Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). The paramagnetic effects generated by these transition metal ions were evaluated by NMR spectroscopy. We show that 2V-8HQ is a rigid and stable transition metal binding tag. The coordination of the metal ion can be assisted by protein sidechains. More importantly, tunable paramagnetic tensors are simply obtained in an α-helix that possesses solvent exposed residues in positions i and i + 3, where i is the residue to be mutated to cysteine, i + 3 is Gln or Glu or i − 4 is His. The coordination of a sidechain carboxylate/amide or imidazole to cobalt(II) results in different structural geometries, leading to different paramagnetic tensors as shown by experimental data.

  10. Site-specific tagging proteins with a rigid, small and stable transition metal chelator, 8-hydroxyquinoline, for paramagnetic NMR analysis

    International Nuclear Information System (INIS)

    Design of a paramagnetic metal binding motif in a protein is a valuable way for understanding the function, dynamics and interactions of a protein by paramagnetic NMR spectroscopy. Several strategies have been proposed to site-specifically tag proteins with paramagnetic lanthanide ions. Here we report a simple approach of engineering a transition metal binding motif via site-specific labelling of a protein with 2-vinyl-8-hydroxyquinoline (2V-8HQ). The protein-2V-8HQ adduct forms a stable complex with transition metal ions, Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). The paramagnetic effects generated by these transition metal ions were evaluated by NMR spectroscopy. We show that 2V-8HQ is a rigid and stable transition metal binding tag. The coordination of the metal ion can be assisted by protein sidechains. More importantly, tunable paramagnetic tensors are simply obtained in an α-helix that possesses solvent exposed residues in positions i and i + 3, where i is the residue to be mutated to cysteine, i + 3 is Gln or Glu or i − 4 is His. The coordination of a sidechain carboxylate/amide or imidazole to cobalt(II) results in different structural geometries, leading to different paramagnetic tensors as shown by experimental data

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

    Science.gov (United States)

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

    2016-01-01

    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. PMID:27694851

  12. Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

    Institute of Scientific and Technical Information of China (English)

    Jun LI; Xue-hao WANG; Xiao-ming WANG; Zhao-lai CHEN

    2006-01-01

    Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-l,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method.First, stable F(ab')2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

  13. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

    Directory of Open Access Journals (Sweden)

    James I. McDonald

    2016-06-01

    Full Text Available Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

  14. TALE homeodomain proteins regulate site-specific terminal differentiation, LCE genes and epidermal barrier.

    Science.gov (United States)

    Jackson, Ben; Brown, Stuart J; Avilion, Ariel A; O'Shaughnessy, Ryan F L; Sully, Katherine; Akinduro, Olufolake; Murphy, Mark; Cleary, Michael L; Byrne, Carolyn

    2011-05-15

    The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). We demonstrate here that a complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. We conclude that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers.

  15. 7 CFR 1726.404 - Non-site specific construction contract closeout.

    Science.gov (United States)

    2010-01-01

    ... UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE ELECTRIC SYSTEM CONSTRUCTION POLICIES AND PROCEDURES Contract Closeout § 1726.404 Non-site specific construction contract closeout. This section is applicable to... 7 Agriculture 11 2010-01-01 2010-01-01 false Non-site specific construction contract...

  16. 77 FR 39234 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2012-07-02

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  17. 76 FR 61350 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2011-10-04

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  18. 76 FR 20651 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2011-04-13

    ... Environmental Management Site-Specific Advisory Board Chairs (76 FR 17118). This notice announces the... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of cancellation of open meeting. SUMMARY: On March 28, 2011, in FR Doc. 2011-7243, on page 17118, the...

  19. 77 FR 55813 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2012-09-11

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

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

    Science.gov (United States)

    2013-05-14

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  1. 76 FR 52944 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-08-24

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of... (EIS) Committee of the Environmental Management Site- Specific Advisory Board (EM SSAB), Nevada. The... environmental restoration, waste management, and related activities. Purpose of the Committee: The purpose...

  2. 76 FR 55370 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-09-07

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of... (EIS) Committee of the Environmental Management Site- Specific Advisory Board (EM SSAB), Nevada. The... environmental restoration, waste management, and related activities. Purpose of the Committee: The purpose...

  3. 78 FR 53135 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2013-08-28

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management,...

  4. 76 FR 63613 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2011-10-13

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  5. 76 FR 80354 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-12-23

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  6. 75 FR 51450 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2010-08-20

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... the Board is to make recommendations ] to DOE-EM and site management in the areas of...

  7. 78 FR 49738 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2013-08-15

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  8. 78 FR 23760 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2013-04-22

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  9. 76 FR 5365 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-01-31

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of... the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  10. 78 FR 20311 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2013-04-04

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of Open Webinar. SUMMARY: This notice announces a webinar of the Environmental Management Site-Specific... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management,...

  11. 77 FR 4027 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2012-01-26

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  12. 75 FR 82004 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2010-12-29

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  13. 76 FR 50204 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-08-12

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of... (EIS) Committee of the Environmental Management Site- Specific Advisory Board (EM SSAB), Nevada. The... environmental restoration, waste management, and related activities. Purpose of the Committee: The purpose...

  14. 76 FR 17118 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2011-03-28

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  15. 75 FR 8050 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2010-02-23

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  16. 75 FR 6018 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2010-02-05

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... the Board is to make recommendations to DOE-EM and site management in the areas of...

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

    Science.gov (United States)

    2013-10-30

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management,...

  18. 77 FR 16021 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2012-03-19

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management,...

  19. 75 FR 65310 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2010-10-22

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  20. 75 FR 13269 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2010-03-19

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  1. 78 FR 26635 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-05-07

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy. ACTION: Notice of... 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. FOR...

  2. 78 FR 61348 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2013-10-03

    ..., 2013 of the Environmental Management Site-Specific Advisory Board, Portsmouth (78 FR 56871). This... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy. ACTION: Notice of Cancellation of Open Meeting. SUMMARY: On September 16, 2013, in FR Doc. 2013-22453, on page...

  3. 75 FR 2859 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2010-01-19

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  4. 78 FR 64208 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2013-10-28

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... environmental restoration, waste management, and related activities. Tentative Agenda Topics: Tuesday,...

  5. 77 FR 12044 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2012-02-28

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... recommendations to DOE-EM and site management in the areas of environmental restoration, waste management,...

  6. 77 FR 28368 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2012-05-14

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental ] Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  7. 78 FR 68431 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2013-11-14

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

  8. 78 FR 4139 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2013-01-18

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... and site management in the areas of environmental restoration, waste management, and...

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

    Science.gov (United States)

    2013-09-25

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... environmental restoration, waste management, and related activities. Tentative Agenda Topics: Wednesday,...

  10. 76 FR 4645 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2011-01-26

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  11. 77 FR 64112 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2012-10-18

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... management in the areas of environmental restoration, waste management, and related activities....

  12. Site-specific properties and irreversible vegetation changes in semi-arid grazing systems

    NARCIS (Netherlands)

    Rietkerk, M; vandenBosch, F; vandeKoppel, J

    1997-01-01

    There is an urgent need to develop a mechanistic understanding of how site-specific properties can lead to irreversible vegetation changes. We show, by means of a bifurcation analysis of two mathematical models, how site-specific properties determine the resilience of vegetation changes in semi-arid

  13. 77 FR 66962 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2012-11-08

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat....

  14. 75 FR 48662 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2010-08-11

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada Test Site. The Federal Advisory Committee Act (Pub. L. 92-463, 86...

  15. 76 FR 21878 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-04-19

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat....

  16. 76 FR 51362 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-08-18

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat....

  17. 76 FR 59393 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-09-26

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat....

  18. 77 FR 75627 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2012-12-21

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat....

  19. 76 FR 28218 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2011-05-16

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific...: Purpose of the Board: The purpose of the Board is to make recommendations to DOE-EM and site management...

  20. 77 FR 16542 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2012-03-21

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific...: Purpose of the Board: The purpose of the Board is to make recommendations to DOE-EM and site management...

  1. 77 FR 51789 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-08-27

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: 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...

  2. 76 FR 29229 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-05-20

    ... Environmental Management Site-Specific Advisory Board, Nevada 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), Nevada. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat....

  3. 76 FR 31599 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2011-06-01

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: 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...

  4. 77 FR 11516 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-02-27

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: 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...

  5. 75 FR 71677 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2010-11-24

    ... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of... the Environmental Management Site-Specific Advisory Board (EM SSAB), Nevada. The Federal Advisory... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  6. 75 FR 19630 - Environmental Management Site-Specific Advisory Board Charter Renewal

    Science.gov (United States)

    2010-04-15

    ... Environmental Management Site-Specific Advisory Board Charter Renewal Pursuant to Section 14(a)(2)(A) of the...-Specific Advisory Board will be renewed for a two-year period beginning April 11, 2010. The Board provides... renewal of the Environmental Management Site- Specific Advisory Board has been determined to be...

  7. A cyclopropene-modified nucleotide for site-specific RNA labeling using genetic alphabet expansion transcription.

    Science.gov (United States)

    Eggert, F; Kath-Schorr, S

    2016-06-01

    Site-specific RNA modification with methyl cyclopropene moieties is performed by T7 in vitro transcription. An existing unnatural base is functionalized with a cyclopropene moiety and used in transcription reactions to produce site-specifically cyclopropene-modified RNA molecules. The posttranscriptional inverse electron demand Diels-Alder cycloaddition reaction with a selected tetrazine-fluorophore conjugate is demonstrated. PMID:27181840

  8. 77 FR 38275 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-06-27

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  9. 76 FR 4644 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

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    2011-01-26

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  10. 76 FR 65190 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-10-20

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  11. 77 FR 23470 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-04-19

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  12. 78 FR 58292 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

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  13. 78 FR 49738 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

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  14. 76 FR 28759 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

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  15. 77 FR 18243 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

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  16. 77 FR 64494 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

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  17. 78 FR 17648 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

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  18. 77 FR 45345 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

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  19. 76 FR 29732 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-05-23

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  20. 75 FR 82001 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-12-29

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  1. 75 FR 24685 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-05-05

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  2. 77 FR 74836 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-12-18

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  3. 76 FR 9572 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-02-18

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  4. 77 FR 2714 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-01-19

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  5. 77 FR 29996 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-05-21

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  6. 78 FR 75552 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2013-12-12

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  7. 76 FR 59393 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-09-26

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  8. 75 FR 13268 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-03-19

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  9. 76 FR 22388 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-04-21

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  10. 76 FR 17637 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-03-30

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  11. 75 FR 51027 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-08-18

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  12. 75 FR 71424 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-11-23

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  13. 78 FR 44942 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2013-07-25

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  14. 75 FR 57462 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-09-21

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  15. 78 FR 63171 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation; Meeting

    Science.gov (United States)

    2013-10-23

    ... Management Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation; Meeting AGENCY: Department...

  16. 77 FR 49442 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-08-16

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  17. 75 FR 65466 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-10-25

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  18. 78 FR 30911 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2013-05-23

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  19. 78 FR 23241 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2013-04-18

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  20. 77 FR 9219 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-02-16

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  1. 75 FR 7576 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-02-22

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  2. 76 FR 78908 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-12-20

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  3. 78 FR 12746 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2013-02-25

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  4. 76 FR 36101 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-06-21

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  5. 78 FR 3890 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2013-01-17

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  6. 76 FR 52944 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-08-24

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  7. 75 FR 3455 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-01-21

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  8. 75 FR 43518 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-07-26

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  9. 75 FR 27998 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-05-19

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  10. 75 FR 35447 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2010-06-22

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  11. 77 FR 58364 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2012-09-20

    ... Site-Specific Advisory Board (EM SSAB), Oak Ridge Reservation. The Federal Advisory Committee Act (Pub... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  12. Assessment of Site Specific Mutational Effect on Transcription Initiation at Escherichia coli Promoter

    Directory of Open Access Journals (Sweden)

    S. Kannan

    2009-01-01

    Full Text Available Problem statement: It is widely accepted thought that the weak promoters control the RNA synthesis and play regulatory role in complex genetic networks in bacterial system. An experiment had been designed to address whether mutations in the -16/-17 region affect the rate of transcription at an activator-independent promoter in E. coli or not? Approach: The aim of this study was to determine whether mutations in the -16/-17 region affect the rate of expression at an activator-dependent promoter in JM109 strain of E. coli. Primers were constructed to amplify the mutant promoter genes through PCR. The amplified PCR product was checked and then inserted into the MCS region of pAA128 plasmid. Further the plasmid vector was transformed into JM109 strain of E. coli and then cloned the selected transformats. Finally, the plasmid from each mutant colony was then sequenced using the protocol supplied with the Amersham Pharmacia Biotech T7 sequencing Kit. The JM109 cultures for which the sequences were determined, then assayed for ß-galactosidase activity to assess the rate of gene expression from the altered promoters. Results: The present investigation revealed that the extended-10 promoter region has a substantial effect on the rate of transcription at weak promoter sequence and also bearing little resemblance to the consensus sequence recognized by RNA. The expression of the genetically engineered plasmid proved that the 2 bps (-16 and -17 base pair found adjacently upstream of the extended-10 promoter have an effect on the level of transcription. This was achieved by site specific base substitutions into the weak promoter of a modified lac operon lacking any activator or repressor binding sites. The results from gene expression assays of several mutants showed a distinct preference for either GG or TT located adjacently upstream of the extended promoter element. Thus the present study emphasized that

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

    Directory of Open Access Journals (Sweden)

    Ding-Pei Long

    Full Text Available 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 have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

  14. Distinct and site-specific phosphorylation of the retinoblastoma protein at serine 612 in differentiated cells.

    Directory of Open Access Journals (Sweden)

    Takayuki Hattori

    Full Text Available The retinoblastoma susceptibility protein (pRB is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.

  15. Site-specific analysis of geothermal development-data files of prospective sites. Vol. III

    Energy Technology Data Exchange (ETDEWEB)

    Williams, F.; Cohen, A.; Pfundstein, R.; Pond, S.

    1978-02-01

    Development scenarios for 37 hydrothermal and geopressured prospects in the United States were analyzed. This third of three volumes presents site-specific data and sample development schedules for the first plant on line at each of the 37 prospects.

  16. 75 FR 82003 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2010-12-29

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... environmental restoration, waste management and related activities. Tentative Agenda: Call to...

  17. 76 FR 8359 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-02-14

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... environmental restoration, waste management and related activities. Tentative Agenda Call to...

  18. 75 FR 7577 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2010-02-22

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... environmental restoration, waste management and related activities. Tentative Agenda: Call to...

  19. 78 FR 10611 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2013-02-14

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... environmental restoration, waste management and related activities. Tentative Agenda Call to...

  20. 76 FR 78909 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-12-20

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... environmental restoration, waste management, and related activities. Tentative Agenda Call to...

  1. Site-specific and geographical segmental social, environmental and ethical disclosures by the Ghanaian mining sector

    OpenAIRE

    Khalid, Sharif.M; Atkins, Jill; Hennell, Alison

    2014-01-01

    Purpose: This paper explores the extent of site-specific and geographic segmental social, environmental and ethical reporting by mining companies operating in Ghana. We aim to: (i) establish a picture of corporate transparency relating to geographic segmentation of social, environmental and ethical reporting which is specific to operating sites and country of operation, and; (ii) gauge the impact of the introduction of integrated reporting on site-specific social, environmental and ethical re...

  2. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Pan [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Xi, Zhaoyong; Wang, Hu [School of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026 (China); Shi, Chaowei [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Xiong, Ying, E-mail: yxiong73@ustc.edu.cn [School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Tian, Changlin, E-mail: cltian@ustc.edu.cn [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China)

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  3. Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

    Science.gov (United States)

    Zhang, Wei-Jie; Luo, Xiao; Liu, Ya-Li; Shao, Xiao-Xia; Wade, John D; Bathgate, Ross A D; Guo, Zhan-Yun

    2012-08-01

    Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  6. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

    Science.gov (United States)

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

    2016-01-22

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

  7. Site-specific water quality guidelines: 1. Derivation approaches based on physicochemical, ecotoxicological and ecological data.

    Science.gov (United States)

    van Dam, R A; Humphrey, C L; Harford, A J; Sinclair, A; Jones, D R; Davies, S; Storey, A W

    2014-01-01

    Generic water quality guidelines (WQGs) are developed by countries/regions as broad scale tools to assist with the protection of aquatic ecosystems from the impacts of toxicants. However, since generic WQGs cannot adequately account for the many environmental factors that may affect toxicity at a particular site, site-specific WQGs are often needed, especially for high environmental value ecosystems. The Australian and New Zealand Guidelines for Fresh and Marine Water Quality provide comprehensive guidance on methods for refining or deriving WQGs for site-specific purposes. This paper describes three such methods for deriving site-specific WQGs, namely: (1) using local reference water quality data, (2) using biological effects data from laboratory-based toxicity testing, and (3) using biological effects data from field surveys. Two case studies related to the assessment of impacts arising from mining operations in northern Australia are used to illustrate the application of these methods. Finally, the potential of several emerging methods designed to assess thresholds of ecological change from field data for deriving site-specific WQGs is discussed. Ideally, multiple lines of evidence approaches, integrating both laboratory and field data, are recommended for deriving site-specific WQGs. PMID:23846952

  8. An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

    Science.gov (United States)

    Kruis, Ilmar C; Löwik, Dennis W P M; Boelens, Wilbert C; van Hest, Jan C M; Pruijn, Ger J M

    2016-09-21

    We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures.

  9. Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain.

    Science.gov (United States)

    Kanje, Sara; von Witting, Emma; Chiang, Samuel C C; Bryceson, Yenan T; Hober, Sophia

    2016-09-21

    Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody's binding to its target. Moreover, an emerging field for therapeutics is antibody-drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.

  10. Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules.

    Science.gov (United States)

    Ahlgren, Sara; Orlova, Anna; Rosik, Daniel; Sandström, Mattias; Sjöberg, Anna; Baastrup, Barbro; Widmark, Olof; Fant, Gunilla; Feldwisch, Joachim; Tolmachev, Vladimir

    2008-01-01

    Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i. PMID:18163536

  11. Development and application of bioassays for a site-specific risk assessment of contaminated soil

    OpenAIRE

    Rila, J.-P.

    2008-01-01

    Soil risk assessment based on generic approaches is accompanied by a large number of uncertainties. In site-specific risk assessment aimed at identifying the actual effects on the ecosystem by using e.g. bioassays in soil elutriates and taking into account land-use these uncertainties can be largely reduced. In this thesis the application and development of bioassays for a site-specific risk assessment of contaminated soil has been discussed. The first part of this thesis deals with the influ...

  12. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors.

    Science.gov (United States)

    Jian, Hui; Wang, Yingwu; Bai, Yan; Li, Rong; Gao, Renjun

    2016-01-01

    Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. PMID:27409601

  13. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

    Directory of Open Access Journals (Sweden)

    Hui Jian

    2016-07-01

    Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

  14. Stable, Site-Specific Fluorescent Tagging Constructs Optimized for Burkholderia Species▿

    OpenAIRE

    Norris, Michael H.; Kang, Yun (Kenneth); Wilcox, Bruce; Hoang, Tung T.

    2010-01-01

    Several vectors that facilitate stable fluorescent labeling of Burkholderia pseudomallei and Burkholderia thailandensis were constructed. These vectors combined the effectiveness of the mini-Tn7 site-specific transposition system with fluorescent proteins optimized for Burkholderia spp., enabling bacterial tracking during cellular infection.

  15. Evaluation of closed-loop site-specific irrigation with wireless sensor network

    Science.gov (United States)

    Automated site-specific sprinkler irrigation system can save water and maximize productivity, but implementing automated irrigation is challenging in system integration and decision making. A controllable irrigation system was integrated into a closed-loop control with a distributed wireless in-fiel...

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

    Science.gov (United States)

    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…

  17. 76 FR 62054 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2011-10-06

    ... Environmental Management Site-Specific Advisory Board Chairs AGENCY: Department of Energy. ACTION: Notice of open teleconference. SUMMARY: This notice announces a teleconference of the Environmental Management... make recommendations to DOE-EM and site management in the areas of environmental restoration,...

  18. 78 FR 36543 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2013-06-18

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE) ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  19. 78 FR 78952 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-12-27

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  20. 76 FR 57981 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-09-19

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  1. 77 FR 59598 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-09-28

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  2. 78 FR 7767 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-02-04

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  3. 76 FR 5147 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2011-01-28

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  4. 77 FR 2282 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-01-17

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy. ACTION: Notice of... of open meeting announcing a meeting on January 19, 2012, of the Environmental Management Site..., 2012, announced in the ] December 23, 2011, issue of the Federal Register (FR Doc. 2011-32913, 76...

  5. 78 FR 73519 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-12-06

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE) ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  6. 77 FR 4799 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-01-31

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental ] Management Site... and site management in the areas of environmental restoration, waste management and related...

  7. 76 FR 80355 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2011-12-23

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... and site management in the areas of environmental restoration, waste management, and...

  8. 78 FR 54460 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-09-04

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

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

    Science.gov (United States)

    2013-03-14

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  10. 75 FR 19379 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2010-04-14

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  11. 75 FR 51026 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2010-08-18

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  12. 75 FR 82002 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2010-12-29

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  13. 76 FR 36100 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-06-21

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy. DOE. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  14. 75 FR 9404 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2010-03-02

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  15. 75 FR 61711 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2010-10-06

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  16. 77 FR 37390 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-06-21

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  17. 75 FR 54600 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2010-09-08

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE) ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  18. 76 FR 70121 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-11-10

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  19. 78 FR 25064 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-04-29

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  20. 76 FR 48148 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2011-08-08

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  1. 77 FR 63300 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-10-16

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  2. 77 FR 50488 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-08-21

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  3. 78 FR 38969 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-06-28

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  4. 77 FR 2283 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-01-17

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  5. 75 FR 24686 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2010-05-05

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  6. 77 FR 43583 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-07-25

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  7. 76 FR 48148 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2011-08-08

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy (DoE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... environmental restoration, waste management, and related activities. Tentative Agenda: Annual Tri-Party...

  8. 77 FR 29997 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-05-21

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  9. 77 FR 31837 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-05-30

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... and site management in the areas of environmental restoration, waste management and related...

  10. 75 FR 66074 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2010-10-27

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  11. 78 FR 45518 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-07-29

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  12. 77 FR 16021 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-03-19

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  13. 78 FR 32640 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2013-05-31

    ... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... management in the areas of environmental restoration, waste management and related activities....

  14. Potentials for site-specific design of MW sized wind turbines

    DEFF Research Database (Denmark)

    Thomsen, K.; Fuglsang, P.; Schepers, G.

    2001-01-01

    The potential for site specific design of MW sized wind turbines is quantified by comparing design loads for wind turbines installed at a range of different sites. The sites comprise on-shore normal flat terrain stand-alone conditions and wind farm conditions together with offshore and mountainou...

  15. Summary of some feasibility studies for site-specific solar industrial process heat

    Energy Technology Data Exchange (ETDEWEB)

    None

    1982-01-01

    Some feasibility studies for several different site specific solar industrial process heat applications are summarized. The followng applications are examined. Leather Tanning; Concrete Production: Lumber and Paper Processing; Milk Processing; Molding, Curing or Drying; Automobile Manufacture; and Food Processing and Preparation. For each application, site and process data, system design, and performance and cost estimates are summarized.

  16. Hellsgate Big Game Winter Range Wildlife Mitigation Site Specific Management Plan for the Hellsgate Project.

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Matthew T.; Judd, Steven L.

    1999-01-01

    This report contains a detailed site-specific management plan for the Hellsgate Winter Range Wildlife Mitigation Project. The report provides background information about the mitigation process, the review process, mitigation acquisitions, Habitat Evaluation Procedures (HEP) and mitigation crediting, current habitat conditions, desired future habitat conditions, restoration/enhancements efforts and maps.

  17. 76 FR 49757 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-08-11

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of Open Meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  18. 76 FR 21878 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2011-04-19

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  19. 77 FR 75626 - Environmental Management Site-Specific Advisory Board, Portsmouth

    Science.gov (United States)

    2012-12-21

    ... Environmental Management Site-Specific Advisory Board, Portsmouth AGENCY: Department of Energy (DOE). ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site... Board is to make recommendations to DOE-EM and site management in the areas of environmental...

  20. MUCOADHESIVE MICROPARTICLES OF CARBOXYMETHYL CHITOSAN FOR SITE SPECIFIC DELIVERY OF PANTOPRAZOLE: FORMULATION AND IN VITRO CHARACTERIZATION

    OpenAIRE

    N.A. Gujarathi*, B.R. Rane and J.K. Patel

    2012-01-01

    Carboxymethyl chitosan, a water soluble modified carboxymethyl substituted chitosan derivative have distinct and unique properties, rendering them effective to form selective permeable mucoadhesive film or membranes. In the formulation of chitosan microsphere an acidic environment is essentially required that may degrade acid sensitive moiety, peptide or protein drugs. Mucoadhesive microparticle of carboxymethyl chitosan was designed and developed for site specific sustained release of Pantop...

  1. Using site-specific soil samples as a substitution for improved hydrological and nonpoint source predictions.

    Science.gov (United States)

    Chen, Lei; Wang, Guobo; Zhong, Yucen; Zhao, Xin; Shen, Zhenyao

    2016-08-01

    Soil databases are one of the most important inputs for watershed models, and the quality of soil properties affects how well a model performs. The objectives of this study were to (1) quantify the sensitivity of model outputs to soil properties and to (2) use site-specific soil properties as a substitution for more accurate hydrological and nonpoint source (H/NPS) predictions. Soil samples were collected from a typical mountainous watershed in China, and the impacts of soil sample parameters on H/NPS predictions were quantified using the Soil and Water Assessment Tool (SWAT). The most sensitive parameters related to predicting flow, sediment, and total phosphorus (TP) mainly were the soil hydrological, the channel erosion processes, and the initial soil chemical environment, respectively. When the site-specific soil properties were used, the uncertainties (coefficient of variation) related to predicting the hydrology, sediment and TP decreased by 75∼80 %, 75∼84 %, and 46∼61 %, respectively. Based on changes in the Nash-Sutcliff coefficient, the model performance improved by 4.9 and 19.45 % for the hydrological and sediment model, accordingly. However, site-specific soil properties did not contribute to better TP predictions because of the high spatial variability of the soil P concentrations across the large watershed. Thus, although site-specific soil samples can be used to obtain more accurate H/NPS predictions, more sampling sites are required to apply this method in large watersheds.

  2. Using site-specific soil samples as a substitution for improved hydrological and nonpoint source predictions.

    Science.gov (United States)

    Chen, Lei; Wang, Guobo; Zhong, Yucen; Zhao, Xin; Shen, Zhenyao

    2016-08-01

    Soil databases are one of the most important inputs for watershed models, and the quality of soil properties affects how well a model performs. The objectives of this study were to (1) quantify the sensitivity of model outputs to soil properties and to (2) use site-specific soil properties as a substitution for more accurate hydrological and nonpoint source (H/NPS) predictions. Soil samples were collected from a typical mountainous watershed in China, and the impacts of soil sample parameters on H/NPS predictions were quantified using the Soil and Water Assessment Tool (SWAT). The most sensitive parameters related to predicting flow, sediment, and total phosphorus (TP) mainly were the soil hydrological, the channel erosion processes, and the initial soil chemical environment, respectively. When the site-specific soil properties were used, the uncertainties (coefficient of variation) related to predicting the hydrology, sediment and TP decreased by 75∼80 %, 75∼84 %, and 46∼61 %, respectively. Based on changes in the Nash-Sutcliff coefficient, the model performance improved by 4.9 and 19.45 % for the hydrological and sediment model, accordingly. However, site-specific soil properties did not contribute to better TP predictions because of the high spatial variability of the soil P concentrations across the large watershed. Thus, although site-specific soil samples can be used to obtain more accurate H/NPS predictions, more sampling sites are required to apply this method in large watersheds. PMID:27146539

  3. 76 FR 11773 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2011-03-03

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463..., Santa Fe, New Mexico 87507. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern New...

  4. 75 FR 53280 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2010-08-31

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463... Pueblo Sur, Taos, New Mexico 87571. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern...

  5. 76 FR 18540 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2011-04-04

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico (known locally as the Northern New Mexico Citizens....m.-4 p.m. ADDRESSES: Holiday Inn Express and Suites, 60 Entrada Drive, Los Alamos, New Mexico...

  6. 77 FR 47047 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2012-08-07

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463... Road, Pojoaque, NM 87506. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern New...

  7. 76 FR 11772 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2011-03-03

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico (known locally as the Northern New Mexico Citizens... Norte, Espanola, New Mexico 87532. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern...

  8. 78 FR 23759 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2013-04-22

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463... Drive, Santa Fe, NM 87501. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern New...

  9. 78 FR 38305 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2013-06-26

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463... 87544. ] FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern New Mexico Citizens'...

  10. Design, development and evaluation of a tree planting-site-specific fumigant applicator

    Science.gov (United States)

    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...

  11. Environmental Restoration Site-Specific Plan for the Portsmouth Gaseous Diffusion Plant, FY 93

    International Nuclear Information System (INIS)

    The purpose of this Site-Specific Plan (SSP) is to describe past, present, and future activities undertaken to implement Environmental Restoration and Waste Management goals at the Portsmouth Gaseous Diffusion Plant (PORTS). The SSP is presented in sections emphasizing Environmental Restoration description of activities, resources, and milestones

  12. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

    NARCIS (Netherlands)

    Witte, Martin D.; Wu, Tongfei; Guimaraes, Carla P.; Theile, Christopher S.; Blom, Annet E. M.; Ingram, Jessica R.; Li, Zeyang; Kundrat, Lenka; Goldberg, Shalom D.; Ploegh, Hidde L.

    2015-01-01

    Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes

  13. Use of GIS-based Site-specific Nitrogen Management for Improving Energy Efficiency

    Science.gov (United States)

    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...

  14. 76 FR 1415 - Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation

    Science.gov (United States)

    2011-01-10

    ... Ridge Reservation. The Federal Advisory Committee Act (Pub. L. 92-463, 86 Stat. 770) requires that... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Oak Ridge Reservation AGENCY: Department of...

  15. Use of GIS-based site-specific nitrogen management for improving energy efficiency

    Science.gov (United States)

    Nitrogen (N) is a significant energy component of in support of crop production but it can be highly variable within fields. To our knowledge, no efforts have been made to employ GIS-based site-specific N management (SSNM) to assess and improve energy costs and efficiency. We examine recent SSNM ca...

  16. Environmental Restoration Site-Specific Plan for the Portsmouth Gaseous Diffusion Plant, FY 93

    Energy Technology Data Exchange (ETDEWEB)

    1993-01-15

    The purpose of this Site-Specific Plan (SSP) is to describe past, present, and future activities undertaken to implement Environmental Restoration and Waste Management goals at the Portsmouth Gaseous Diffusion Plant (PORTS). The SSP is presented in sections emphasizing Environmental Restoration description of activities, resources, and milestones.

  17. Software Design for Wireless Sensor-based Site-specific Irrigation

    Science.gov (United States)

    In-field sensor-based site-specific irrigation management is of benefit to producers for efficient water management. Integration of the decision making process with the controls is a viable option for determining when and where to irrigate, and how much water to apply. This research presents the des...

  18. Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

    Science.gov (United States)

    Taylor, John-Stephen

    2015-12-01

    How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

  19. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

    DEFF Research Database (Denmark)

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

    2014-01-01

    have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...

  20. How much can we say about site-specific cancer radiation risks?

    Science.gov (United States)

    Preston, D L; Krestinina, L Yu; Sokolnikov, M E; Ron, E; Davis, F G; Ostroumova, E V; Gilbert, E S

    2010-12-01

    Studies of Mayak workers and people who lived along the Techa River have demonstrated significant associations between low-dose-rate radiation exposure and increased solid cancer risk. It is of interest to use the long-term follow-up data from these cohorts to describe radiation effects for specific types of cancer; however, statistical variability in the site-specific risk estimates is large. The goal of this work is to describe this variability and provide Bayesian adjusted risk estimates. We assume that the site-specific estimates can be viewed as a sample from some underlying distribution and use Bayesian methods to produce adjusted excess relative risk per gray estimates in the Mayak and Techa River cohorts. The impact of the adjustment is compared to that seen in similar analyses in the atomic bomb survivors. Site-specific risk estimates in the Mayak and Techa River cohorts have large uncertainties. Unadjusted estimates vary from implausibly large decreases to large increases, with a range that greatly exceeds that found in the A-bomb survivors. The Bayesian adjustment markedly reduced the range of the site-specific estimates for the Techa River and Mayak studies. The extreme variability in the site-specific risk estimates is largely a consequence of the small number of excess cases. The adjusted estimates provide a useful perspective on variation in the actual risks. However, additional work on interpretation of the adjusted estimates, extension of the methods used in describing effect modification, and making more use of prior knowledge is needed to make these methods useful. PMID:21128806

  1. In vivo tumor angiogenesis imaging with site-specific labeled {sup 99m}Tc-HYNIC-VEGF

    Energy Technology Data Exchange (ETDEWEB)

    Blankenberg, Francis G. [Stanford University, Division of Nuclear Medicine/Department of Radiology and MIPS (Molecular Imaging Program at Stanford), Stanford, CA (United States); Stanford University, Department of Pediatrics, Stanford, CA (United States); Backer, Marina V.; Patel, Vimalkumar; Backer, Joseph M. [SibTech, Inc., Newington, CT (United States); Levashova, Zoia [Stanford University, Division of Nuclear Medicine/Department of Radiology and MIPS (Molecular Imaging Program at Stanford), Stanford, CA (United States)

    2006-07-15

    We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test {sup 99m}Tc-HYNIC-C-tagged VEGF ({sup 99m}Tc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. {sup 99m}Tc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200-300 {mu}Ci, 1-2 {mu}g protein) followed by microSPECT imaging 1 h later. Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with {sup 99m}Tc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3{+-}5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o.d. x 4; 1.14{+-}0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. x 7; 1.03{+-}0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of {sup 99m}Tc/biotin-inactivated VEGF, as compared with {sup 99m}Tc-HYNIC-VEGF. {sup 99m}Tc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. {sup 99m}Tc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy. (orig.)

  2. Forster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.

    Directory of Open Access Journals (Sweden)

    James D Fessenden

    Full Text Available BACKGROUND: While the static structure of the intracellular Ca(2+ release channel, the ryanodine receptor type 1 (RyR1 has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes. METHODOLOGY/PRINCIPAL FINDINGS: A novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni(2+/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP. GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm. Cy3NTA bound to His(10-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His(10 tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His(10 tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other. CONCLUSIONS/SIGNIFICANCE: These findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1

  3. Site-specific electron-induced cross-linking of ortho-carborane to form semiconducting boron carbide

    Energy Technology Data Exchange (ETDEWEB)

    Pasquale, Frank L. [Department of Chemistry, University of North Texas, Denton, TX 76203 (United States); Kelber, Jeffry A., E-mail: kelber@unt.edu [Department of Chemistry, University of North Texas, Denton, TX 76203 (United States)

    2012-01-15

    Semiconducting boron carbide (B{sub 10}C{sub 2}H{sub x}) films have been formed by bombardment of condensed ortho-carborane (closo-1,2-dicarbadodecaborane) multilayers on polycrystalline copper substrates by 200 eV electrons under ultra-high vacuum conditions. The film formation process was characterized by X-ray and ultraviolet photoelectron spectroscopies. Electron bombardment results in the cross-linking of the icosahedral units. The cross-linking is accompanied by a shift in the B(1s) binding energy indicating site-specific cross-linking between two boron sites on adjacent carborane icosahedra. An additional shift in valence band binding energies attributed to the surface photovoltage effect is indicative of the formation of a p-type semiconductor. This is the first report of B{sub 10}C{sub 2}H{sub x} formation by electron bombardment of condensed films, and the data indicate that this method is a viable route towards formation of ultra-thin films of tailored composition and cross-linkages for emerging nanoelectronics and sensor applications.

  4. Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins

    DEFF Research Database (Denmark)

    Lambertsen, L.; Sternberg, Claus; Molin, Søren

    2004-01-01

    The mini-Tn7 transposon system is a convenient tool for site-specific tagging of bacteria in which the tagging DNA is inserted at a unique and neutral chromosomal site. We have expanded the panel of mini-Tn7 delivery plasmids expressing different fluorescent proteins (stable and unstable) from...... the Escherichia coli lac derived promoter, P-A1/04/03, or from the growth-rate-dependent Escherichia coli promoter P-rrnB P1. The mini-Tn7 transposons were inserted and tested in the soil bacterium, Pseudomonas putida KT2440. Successful and site-specific tagging was verified by Southern blots as well as by PCR....... Furthermore, the effect of fluorescent protein expression on the cellular growth rate was tested by growth competition assays....

  5. Proposed Site-Specific Response Spectra for Surabaya-Madura Bridge

    Directory of Open Access Journals (Sweden)

    Dyah Kusumastuti

    2008-01-01

    Full Text Available This paper presents a site-specific seismic hazard study to determine the recommended seismic design criteria for Suramadu Bridge. The study is performed using probabilistic seismic hazard approach to determine maximum acceleration and response spectra at bedrock and followed by local site effect analysis to determine maximum acceleration and response spectra at ground surface. The probabilistic seismic hazard analysis (PSHA is carried out using 3-dimension (3-D seismic source models (fault source model. Two hazard levels are analysed to represent 150 and 3,300 years return period of ground motion around site location. The local site effect analysis is performed using 1-dimension (1-D shear wave propagation theory to obtain peak ground acceleration and response spectra at ground surface. Finally, the site-specific surface response spectra with 5 percent damping are developed based on the mean plus one standard deviation concept from the result of local site effect analysis.

  6. Development of a site specific environmental radiation assessment model for Youngkwang NPPs

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Yang Geun; Song, Myung Jae; Shon, Soon Hwan; Shin, Sang Woon; Lee, Gap Bock; Yang, Kyung Hwa [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Seong Pyung; Kim, Jeong Kyu; Shin, Dae Yoon; Kim, Hyun Ku; Lee, Kyung Jin [Chosun University, Kwangju (Korea, Republic of)

    1996-12-31

    During the normal operation of nuclear power plants, a very small amount of radionuclide materials is released to the environment. The radionuclide releases to the environment may give rise to some additional radiation dose through a number of pathways. With a few exception, radionuclide transports are highly dependent on the environmental conditions such as topological features, atmospheric and oceanic conditions and socio-environmental characteristics, etc. The dose calculation in Korea are presently being performed using the models in US NRC Reg. Guide. However, these models which were originally released by the US NRC in 1977 are inadequate to deal with environmental movement of radionuclides in consideration of complex terrain, tidal condition of the Yellow Sea, and Youngkwang site specific conditions. KEPRI has developed the models to consider the site specific characteristics around Youngkwang NPPs, to give the realistic dose assessment, and to improve assurance and reliability in public dose calculation. (author). 41 figs., 70 refs.

  7. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2007-02-21

    Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

  8. Site-specific growth of Au particles on ZnO nanopyramids under ultraviolet illumination

    KAUST Repository

    Yao, Kexin

    2011-01-01

    In this work, wurtzite ZnO nanocrystals with unique "pyramid" morphology were firstly prepared via solvothermal synthesis. It was determined that the ZnO nanopyramids are grown along the polar c-axis with the vertexes pointing to the [001] direction. When the mixture of ZnO nanopyramids and Au precursor (HAuCl4) was exposed to ultraviolet (UV) illumination, Au particles were site-specifically formed on the vertexes of ZnO nanopyramids. The obtained Au/ZnO nanocomposite showed significantly enhanced photocatalytic activity as compared to the bare ZnO nanopyramids. First-principles based calculations well explained the formation of ZnO nanopyramids as well as the site-specific growth of Au, and revealed that during the photocatalysis process the Au particles can accommodate photoelectrons and thus facilitate the charge separation. © 2011 The Royal Society of Chemistry.

  9. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish

    Directory of Open Access Journals (Sweden)

    Atsuo Kawahara

    2016-05-01

    Full Text Available 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.

  10. Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9

    OpenAIRE

    Junfeng Jiang; Li Zhang; Xingliang Zhou; Xi Chen; Guanyi Huang; Fengsheng Li; Ruizhe Wang; Nancy Wu; Youzhen Yan; Chang Tong; Sankalp Srivastava; Yue Wang; Houqi Liu; Qi-Long Ying

    2016-01-01

    Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility, and cancers. The lack of cellular and animal models for chromosomal translocations, however, has hampered our ability to understand the underlying disease mechanisms and to develop new therapies. Here, we show that site-specific chromosomal translocations can be generated in mouse embryonic stem cells (mESCs) via CRISPR/Cas9. Mouse ESCs carrying ...

  11. Adding value to spatially managed inputs by understanding site-specific yield response

    OpenAIRE

    Bullock, David S.; Lowenberg-DeBoer, Jess; Swinton, Scott M.

    2002-01-01

    Many mechanized crop producers and agribusinesses are fascinated with precision agriculture technology, but adoption has lagged behind the expectations. Among the reasons for slow adoption of precision agriculture technology is that initial users focused excessively on in-field benefits from variable-rate fertilizer application using regional average fertilizer recommendations. This article illustrates how greater use of site-specific crop response information can improve variable rate input ...

  12. Multiple new site-specific recombinases for use in manipulating animal genomes

    OpenAIRE

    Nern, Aljoscha; Pfeiffer, Barret D.; Svoboda, Karel; Rubin, Gerald M.

    2011-01-01

    Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast—KD, B2, B3, and R—are highly active and nontoxic in Drosophila and that...

  13. Site-Specific Waste Management Instruction - 100-DR-1 Group 2 Sites

    International Nuclear Information System (INIS)

    This site-specific waste management instruction (SSWMI) provides guidance for the management of wastes that may be generated during the excavation and remediation of the 100-DR-1 Group 2 sites. The management of waste generated as a result of these activities will be as directed in this SSWMI. This SSWMI will be revised to incorporate guidance for management of wastes encountered that are not addressed in this SSWMI

  14. Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I.

    OpenAIRE

    O'Connor, D; Stöhrer, G

    1985-01-01

    Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and "nick-mers", conditions of single-strand-directed DNA synthesis and nick-translation could be created. Our results show that the polymerase can bypass bot...

  15. Proposed Site-Specific Response Spectra for Surabaya-Madura Bridge

    OpenAIRE

    Dyah Kusumastuti; R. Bambang Boediono; Hendriyawan Hendriyawan; Masyhur Irsyam; Donny T. Dangkua; Engkon K. Kertapati

    2008-01-01

    This paper presents a site-specific seismic hazard study to determine the recommended seismic design criteria for Suramadu Bridge. The study is performed using probabilistic seismic hazard approach to determine maximum acceleration and response spectra at bedrock and followed by local site effect analysis to determine maximum acceleration and response spectra at ground surface. The probabilistic seismic hazard analysis (PSHA) is carried out using 3-dimension (3-D) seismic source models (fault...

  16. Site-specific antibodies to human erythropoietin directed toward the NH2-terminal region.

    OpenAIRE

    Sue, J M; Sytkowski, A. J.

    1983-01-01

    Site-specific antibodies to human erythropoietin have been raised in rabbits immunized with a synthetic polypeptide composed of the putative 26 NH2-terminal amino acids of the hormone. The immunogenic peptide was coupled to bovine serum albumin. Antibodies specific for peptide were detected by enzyme-linked immunosorbent assay. They immunoprecipitated both highly purified 125I-labeled erythropoietin and biologically active erythropoietin. The immunoprecipitation of 125I-labeled erythropoietin...

  17. Improving nitrogen fertilization in rice by site-specific N management. A review

    OpenAIRE

    Peng, Shaobing; Buresh, Roland J.; Huang, Jianliang; Zhong, Xuhua; Zou, Yingbin; Yang, Jianchang; Wang, Guanghuo; Liu, Yuanying; Hu, Ruifa; Tang, Qiyuan; Cui, Kehui; Zhang, Fusuo; Dobermann, Achim

    2010-01-01

    Excessive nitrogen (N) application to rice (Oryza sativa L.) crop in China causes environmental pollution, increases the cost of rice farming, reduces grain yield and contributes to global warming. Scientists from the International Rice Research Institute have collaborated with partners in China to improve rice N fertilization through site-specific N management (SSNM) in China since 1997. Field experiments and demonstration trials were conducted initially in Zhejiang province and gradually ex...

  18. Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

    OpenAIRE

    Padan, E; Sarkar, H K; Viitanen, P V; Poonian, M S; Kaback, H R

    1985-01-01

    The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a ...

  19. Programmable Site-Specific Nucleases for Targeted Genome Engineering in Higher Eukaryotes.

    Science.gov (United States)

    Govindan, Ganesan; Ramalingam, Sivaprakash

    2016-11-01

    Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site-specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site-specific genomic double-strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error-prone non-homologous end joining (NHEJ) resulting in gene inactivations/knockouts. Alternatively, the DSB can be repaired through homology-directed repair (HDR) using a donor DNA template, which leads to the introduction of desired sequence modifications at the predetermined site. Here, we summarize the role of three classes of nucleases; zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system in achieving targeted genome modifications. Further, we discuss the progress towards the applications of programmable site-specific nucleases (SSNs) in treating human diseases and other biological applications in economically important higher eukaryotic organisms such as plants and livestock. J. Cell. Physiol. 231: 2380-2392, 2016. © 2016 Wiley Periodicals, Inc. PMID:26945523

  20. Site-specific unfolding thermodynamics of a helix-turn-helix protein.

    Science.gov (United States)

    Amunson, Krista E; Ackels, Loren; Kubelka, Jan

    2008-07-01

    The thermal unfolding of a 40-residue helix-turn-helix subdomain of the P22 viral coat protein was investigated using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) with site-specific 13C isotopic labeling. Helix-turn-helix is the simplest alpha-helical structural motif that combines both secondary and tertiary structural elements. The CD of individual helical fragments reveals that the P22 subdomain is stabilized by tertiary interhelical interactions. Overall the temperature-dependent CD and FTIR data can be described by a three-state process with a partially folded intermediate. However, the analysis of the site-specific 13C IR signals reveals distinct unfolding thermodynamics for each of the labeled sites. The thermodynamic parameters of the thermal unfolding of each of the labeled segments were obtained using singular value decomposition in combination with target transformation and global fitting. The P22 subdomain unfolds from the N-terminus toward the helical segments near the turn. Our results show that as few as two 13C labeled residues can be detected in a 40 residue protein and provide local, site-specific structural information about protein unfolding, which is not resolved by standard, nonsite-specific spectroscopic probes.

  1. Site-specific estimates of water yield applied in regional acid sensitivity surveys across western Canada

    Directory of Open Access Journals (Sweden)

    Patrick D. SHAW

    2010-08-01

    Full Text Available Runoff or water yield is an important input to the Steady-State Water Chemistry (SSWC model for estimating critical loads of acidity. Herein, we present site-specific water yield estimates for a large number of lakes (779 across three provinces of western Canada (Manitoba, Saskatchewan, and British Columbia using an isotope mass balance (IMB approach. We explore the impact of applying site-specific hydrology as compared to use of regional runoff estimates derived from gridded datasets in assessing critical loads of acidity to these lakes. In general, the average water yield derived from IMB is similar to the long-term average runoff; however, IMB results suggest a much larger range in hydrological settings of the lakes, attributed to spatial heterogeneity in watershed characteristics and landcover. The comparison of critical loads estimates from the two methods suggests that use of average regional runoff data in the SSWC model may overestimate critical loads for the majority of lakes due to systematic skewness in the actual runoff distributions. Implications for use of site-specific hydrology in regional critical loads assessments across western Canada are discussed.

  2. Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene- Deoxyadenosine Lesions

    Science.gov (United States)

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

    Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. PMID:26344227

  3. Identification of Site-Specific Stroke Biomarker Candidates by Laser Capture Microdissection and Labeled Reference Peptide

    Directory of Open Access Journals (Sweden)

    Tingting Lian

    2015-06-01

    Full Text Available The search to date for accurate protein biomarkers in acute ischemic stroke has taken into consideration the stage and/or the size of infarction, but has not accounted for the site of stroke. In the present study, multiple reaction monitoring using labeled reference peptide (LRP following laser capture microdissection (LCM is used to identify site-specific protein biomarker candidates. In middle cerebral artery occlusion (MCAO rat models, both intact and infarcted brain tissue was collected by LCM, followed by on-film digestion and semi-quantification using triple-quadrupole mass spectrometry. Thirty-four unique peptides were detected for the verification of 12 proteins in both tissue homogenates and LCM-captured samples. Six insoluble proteins, including neurofilament light polypeptide (NEFL, alpha-internexin (INA, microtubule-associated protein 2 (MAP2, myelin basic protein (MBP, myelin proteolipid protein (PLP and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP, were found to be site-specific. Soluble proteins, such as neuron-specific enolase (NSE and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1, and some insoluble proteins, including neurofilament heavy polypeptide (NEFH, glial fibrillary acidic protein (GFAP, microtubule-associated protein tau (MAPT and tubulin β-3 chain (TUBB3, were found to be evenly distributed in the brain. Therefore, we conclude that some insoluble protein biomarkers for stroke are site-specific, and would make excellent candidates for the design and analysis of relevant clinical studies in the future.

  4. Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases.

    Science.gov (United States)

    Raeeszadeh-Sarmazdeh, Maryam; Parthasarathy, Ranganath; Boder, Eric T

    2015-04-01

    We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.

  5. Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

    Directory of Open Access Journals (Sweden)

    Weis Robert M

    2005-03-01

    Full Text Available Abstract Background Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr, which contributes to an increase in the steady-state (adapted methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA and an adaptor protein (CheW, but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. Results Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493. The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively differed from those found previously for the aspartate receptor (Tar, which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately were the largest at site one, ~3 and 6-fold, respectively, and the least at site four, no change and ~2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four. This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. Conclusion The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of

  6. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    Science.gov (United States)

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  7. A Tissue Engineering Approach to Site-Specific Reconstruction of Skeletal Structures of the Maxillofacial Region:Part Ⅱ

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ [See Part I in Shanghai J Stomatol 9(1):34-38,2000] III. An Image-Based Approach to Design and Manufacture of a Site-Specific Bone/Cartilage Construct Our image-based approach to the design and manufacture of a site-specific scaffold for maxillofacial reconstruction consists of 5 basic steps (Figure 2).

  8. Char-recirculation biomass gasification system--a site-specific feasibility study

    International Nuclear Information System (INIS)

    A site-specific feasibility study was conducted for a char-recirculation biomass gasification plant which would dispose of the chippable solid residues of the area sawmills. The plant would receive green hardwood chips and convert them into active charcoal while producing process steam and electrical power. An economic analysis was performed on the basis of not-for-profit operation, marketing crushed active charcoal to a broker at a discounted price, and displacing purchased electric power. Given a market for the active charcoal, the plant was judged to be economically viable

  9. Site-specific growth of Au particles on ZnO nanopyramids under ultraviolet illumination

    Science.gov (United States)

    Yao, Ke Xin; Liu, Xin; Zhao, Lan; Zeng, Hua Chun; Han, Yu

    2011-10-01

    In this work, wurtzite ZnO nanocrystals with unique ``pyramid'' morphology were firstly prepared via solvothermal synthesis. It was determined that the ZnO nanopyramids are grown along the polar c-axis with the vertexes pointing to the [001] direction. When the mixture of ZnO nanopyramids and Au precursor (HAuCl4) was exposed to ultraviolet (UV) illumination, Au particles were site-specifically formed on the vertexes of ZnO nanopyramids. The obtained Au/ZnO nanocomposite showed significantly enhanced photocatalytic activity as compared to the bare ZnO nanopyramids. First-principles based calculations well explained the formation of ZnO nanopyramids as well as the site-specific growth of Au, and revealed that during the photocatalysis process the Au particles can accommodate photoelectrons and thus facilitate the charge separation.In this work, wurtzite ZnO nanocrystals with unique ``pyramid'' morphology were firstly prepared via solvothermal synthesis. It was determined that the ZnO nanopyramids are grown along the polar c-axis with the vertexes pointing to the [001] direction. When the mixture of ZnO nanopyramids and Au precursor (HAuCl4) was exposed to ultraviolet (UV) illumination, Au particles were site-specifically formed on the vertexes of ZnO nanopyramids. The obtained Au/ZnO nanocomposite showed significantly enhanced photocatalytic activity as compared to the bare ZnO nanopyramids. First-principles based calculations well explained the formation of ZnO nanopyramids as well as the site-specific growth of Au, and revealed that during the photocatalysis process the Au particles can accommodate photoelectrons and thus facilitate the charge separation. Electronic supplementary information (ESI) available: TEM images of Au/ZnO nanocomposites with Au nanoparticles in different sizes, density of states of Au55 and Au20 deposited on ZnO (001) surface, and the first-principles calculation details. See DOI: 10.1039/c1nr10685f

  10. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna W.

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  11. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    Science.gov (United States)

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  12. Site Specific Advisory Board initiative, evaluation survey results supplementary appendix: Summary of individual site results

    International Nuclear Information System (INIS)

    This Appendix presents results of the Site-Specific Advisory Board (SSAB) Initiative for each of the 11 sites that participated in the survey. These individual results are a supplement to the June 1996 Summary Report which presented overall survey results. Results are presented in 11 sections, arranged alphabetically by site. Each section includes a series of figures and tables that parallel those presented in the Summary Report. To facilitate comparison, figures are presented both for the individual site and for the overall long survey. The sequence of sections is: Fernald, Hanford, Idaho, Los Alamos, Monticello, Nevada, Pantex, Rocky Flats, St. Louis, Sandia, and Savannah River

  13. Site specific analysis of geothermal development-data files of prospective sites. Volume II

    Energy Technology Data Exchange (ETDEWEB)

    Trehan, R.; Cohen, A.; Gupta, J.; Jacobsen, W.; Leigh, J.; True, S.

    1978-08-01

    Development scenarios for 37 hydrothermal and geopressured prospects in the United States were analyzed to assist DOE's Division of Geothermal Energy in mission-oriented planning of geothermal resource development. This second volume of the three-volume series contains the detailed site-specific analyses in terms of technological, economic, and other requirements for meeting the postulated schedules. This presentation should be used in conjunction with Volume III, which contains detailed descriptive data files for each of the 37 prospects. These data files were used for the analyses contained in Volume II and should be useful for other geothermal resource studies. (JGB)

  14. Toward site-specific, homogeneous and highly stable fluorescent silver nanoclusters fabrication on triplex DNA scaffolds

    OpenAIRE

    Feng, Lingyan; Huang, Zhenzhen; Ren, Jinsong; Qu, Xiaogang

    2012-01-01

    A new strategy to create site-specific, homogeneous, and bright silver nanoclusters (AgNCs) with high-stability was demonstrated by triplex DNA as template. By reasonable design of DNA sequence, homogeneous Ag2 cluster was obtained in the predefined position of CG.C+ site of triplex DNA. This strategy was also explored for controlled alignment of AgNCs on the DNA nanoscaffold. To the best of our knowledge, this was the first example to simultaneously answer the challenges of excellent site-sp...

  15. Criteria for compilation of a site-specific thermodynamic database for geochemical speciation calculations

    International Nuclear Information System (INIS)

    A methodology has been developed to establish a site-specific database appropriate to geochemical modelling the critical components and the wide range of near field conditions expected in the low level radioactive waste disposal site at Drigg in the UK. Several databases available in the public domain have been compared to select a foundation database. The foundation database was 'trimmed-down' and then customised to suit Drigg applications. The species dominant at Drigg have been identified and the thermodynamic constants of these species have been critically evaluated. The evaluated database has been validated for quality by comparing speciation calculations with plutonium and uranium experimental solubility results. (orig.)

  16. Precision nitrogen management across site-specific management zones in irrigated maize production systems

    Science.gov (United States)

    Inman, Daniel Joseph, II

    In the United States, crop nitrogen-use efficiency (NUE) is very low. Approximately 33% of all N applied towards cereal crop production is captured in the harvested grain. Precision agricultural practices have shown potential for increasing crop NUE. Objectives were: (i) to characterize the within field spatial variability of N uptake across irrigated corn production fields, (ii) to quantify and compare N uptake and grain yield across three site specific management zones (SSMZs), (iii) to compare grain yield response to applied N between management zones, (iv) to examine the relationships between normalized difference vegetation index (NDVI) determined early in the growing season, site-specific management zones, and relative maize yield; (v) to determine if NDVI can be used to estimate relative maize yield; (vi) to determine if site-specific management zones can be used in conjunction with remote sensing to provide yield estimates in irrigated maize, and (vii) to evaluate the effectiveness of using a hand-held active remote sensing instrument to estimate yield potential in irrigated maize. This study was conducted on commercially-operated irrigated production maize fields throughout northeastern Colorado. For objectives i, ii, and iii, fields were classified into high, medium, and low site specific management zones. For objective iv, v, and vi, aerial imagery was acquired at approximately the eight-leaf crop growth stage. Grain was harvested using a commercial-combine outfitted with a yield monitor at the crop's physiological maturity. Objective iv was analyzed using percent areal agreement, kappa statistics, and regression analysis. Objectives v and vi were analyzed using regression analysis with cross-validation and indicator variables. For objective vii, the GreenSeeker(TM) active remote sensing unit was used to measure red and near infrared reflectance of the crop canopy. A response index (RI) was calculated the ratio of the reflectance of an area of interest

  17. Environmental restoration and waste management Site-Specific Plan for the Oak Ridge Reservation

    International Nuclear Information System (INIS)

    The United States Department of Energy (DOE) is committed to achieving and maintaining environmental regulatory compliance while responding to public concerns and emphasizing waste minimization. DOE publishes the Environmental Restoration and Waste Management Five-Year Plan (FYP) annually to document its progress towards these goals. The purpose of this Site-Specific Plan (SSP) is to describe the activities undertaken to implement the FYP goals at the DOE Oak Ridge Field Office (DOE/OR) installations and programs specifically for the Oak Ridge Reservation (ORR) and surrounding areas. This SSP addresses activities and goals to be accomplished during FY93 even through the FYP focuses on FY94

  18. Environmental restoration and waste management Site-Specific Plan for the Oak Ridge Reservation. FY 1993

    Energy Technology Data Exchange (ETDEWEB)

    1993-01-15

    The United States Department of Energy (DOE) is committed to achieving and maintaining environmental regulatory compliance while responding to public concerns and emphasizing waste minimization. DOE publishes the Environmental Restoration and Waste Management Five-Year Plan (FYP) annually to document its progress towards these goals. The purpose of this Site-Specific Plan (SSP) is to describe the activities undertaken to implement the FYP goals at the DOE Oak Ridge Field Office (DOE/OR) installations and programs specifically for the Oak Ridge Reservation (ORR) and surrounding areas. This SSP addresses activities and goals to be accomplished during FY93 even through the FYP focuses on FY94.

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

    Science.gov (United States)

    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. PMID:27502111

  20. Impairments in site-specific AS160 phosphorylation and effects of exercise training

    DEFF Research Database (Denmark)

    Consitt, Leslie A; Van Meter, Jessica; Newton, Christopher 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 lifespan (18 to 84 years) and if endurance- and/or strength-oriented exercise training ...... population and that exercise training is an effective intervention for treating these impairments.......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 lifespan (18 to 84 years) and if endurance- and/or strength-oriented exercise training...... 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 (PAS), but not Ser-318 or Ser-751. Twelve weeks of either endurance- or strength-oriented exercise training increased whole...

  1. Environmental Restoration and Waste Management Site-Specific Plan (SSP) for fiscal year 1992 (FY92)

    International Nuclear Information System (INIS)

    The FY-92 Site-Specific Plan (FY-92 SSP) for environmental restoration and waste management at the Idaho National Engineering Laboratory (INEL) is designed to provide the reader with easy access to the status of environmental restoration and waste management activities at INEL. The first chapter provides background on INIEL's physical environment, site history and mission, and general information about the site and its facilities. In addition, this chapter discusses the inter-relationships between the Site Specific Plan, the Environmental Restoration and Waste Management Five-Year Plan, the environmental restoration and waste management prioritization systems, and the Activity Data Sheets (ADSs) for environmental restoration and waste management. This discussion should help readers understand what the SSP is and how it fits into the environmental restoration and waste management process at INEL. This understanding should provide the reader with a better context for understanding the discussions in the SSP as well as a better feel for how and what to comment on during the public comment period that will be held from the first of September through the end of October 1991

  2. Applying Nitrogen Site-Specifically Using Soil Electrical Conductivity Maps and Precision Agriculture Technology

    Directory of Open Access Journals (Sweden)

    E.D. Lund

    2001-01-01

    Full Text Available Soil texture varies significantly within many agricultural fields. The physical properties of soil, such as soil texture, have a direct effect on water holding capacity, cation exchange capacity, crop yield, production capability, and nitrogen (N loss variations within a field. In short, mobile nutrients are used, lost, and stored differently as soil textures vary. A uniform application of N to varying soils results in a wide range of N availability to the crop. N applied in excess of crop usage results in a waste of the grower’s input expense, a potential negative effect on the environment, and in some crops a reduction of crop quality, yield, and harvestability. Inadequate N levels represent a lost opportunity for crop yield and profit. The global positioning system (GPS-referenced mapping of bulk soil electrical conductivity (EC has been shown to serve as an effective proxy for soil texture and other soil properties. Soils with a high clay content conduct more electricity than coarser textured soils, which results in higher EC values. This paper will describe the EC mapping process and provide case studies of site-specific N applications based on EC maps. Results of these case studies suggest that N can be managed site-specifically using a variety of management practices, including soil sampling, variable yield goals, and cropping history.

  3. Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

    Directory of Open Access Journals (Sweden)

    Rie Kawai

    2012-03-01

    Full Text Available Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienylimidazo[4,5-b]pyridine (Ds and pyrrole-2-carbaldehyde (Pa functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

  4. ADMA/SDMA in Elderly Subjects with Asymptomatic Carotid Atherosclerosis: Values and Site-Specific Association

    Directory of Open Access Journals (Sweden)

    Graziano Riccioni

    2014-04-01

    Full Text Available Asymmetric dimethylarginine (ADMA is an endogenous nitric oxide synthase (NOS inhibitor known as a mediator of endothelial dysfunction and atherosclerosis. Circulating ADMA levels are correlated with cardiovascular risk factors such as hypercholesterolemia, arterial hypertension, diabetes mellitus, hyperhomocysteinemia, age and smoking. We assessed the relationship between ADMA values and site-specific association of asymptomatic carotid atherosclerosis (intima-media thickness (CIMT and plaque in elderly subjects. One hundred and eighty subjects underwent a complete history and physical examination, determination of serum chemistries and ADMA levels, and carotid ultrasound investigation (CUI. All subjects had no acute or chronic symptoms of carotid atherosclerosis. Statistical analyses showed that high plasma levels of ADMA/SDMA were positively correlated to carotid atherosclerosis (CIMT and plaque (p < 0.001, with significant site-specific association. Total cholesterol, low density lipoprotein cholesterol, triglycerides and C-reactive protein plasma concentrations were significantly associated with asymptomatic carotid atherosclerosis (p < 0.001. High serum concentrations of ADMA and SDMA were associated with carotid atherosclerotic lesions as measured by CIMT ad plaque and may represent a new marker of asymptomatic carotid atherosclerosis in elderly subjects.

  5. Testing the atmospheric dispersion model of CSA N288.1 with site-specific data

    Energy Technology Data Exchange (ETDEWEB)

    Chouhan, S.L.; Davis, P.A

    2001-01-01

    The atmospheric dispersion component of CSA Standard N288. 1, which provides guidelines for calculating derived release limits, has been tested. Long-term average concentrations of tritium in air were predicted using site-specific release rates and meteorological data and compared with measured concentrations at 43 monitoring sites at all CANDU stations in Canada. The predictions correlate well with the observations but were found to be conservative, overestimating by about 50% on average. The model overpredicted 84% of the time, with the highest prediction lying a factor of 5.5 above the corresponding observation. The model underpredicted the remaining 16% of the time, with the lowest prediction about one-half of the corresponding measurement. Possible explanations for this bias are discussed but no single reason appears capable of accounting for the discrepancy. Rather, the tendency to overprediction seems to result from the cumulative effects of a number of small conservatisms in the model. The model predictions were slightly better when site-specific meteorological data were used in the calculations in place of the default data of N288.1. Some large discrepancies between predictions and observations at specific monitoring sites suggest that it is the measurements rather than the model that are at fault. The testing has therefore provided a check on the observations as well as on the model. Recommendations on model use and data collection are made to improve the level of agreement between predictions and observations in the future. (author)

  6. New developments for the site-specific attachment of protein to surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2005-05-12

    Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

  7. Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG₁.

    Directory of Open Access Journals (Sweden)

    Feifan Yu

    Full Text Available Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁. Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs. The best variant, denoted Z(F5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe

  8. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

    Directory of Open Access Journals (Sweden)

    Rajeevan Mangalathu S

    2009-06-01

    Full Text Available Abstract Background Differential expression of perforin (PRF1, a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction. Findings We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA or DNA extracted from peripheral blood mononuclear cells (PBMC DNA from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18% to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92% in the distal enhancer region (CpG sites 1–10, a variable methylation (range 49%–83% in the methylation sensitive region (CpG sites 11–17, and a progressively declining methylation level (range 12%–80% in the proximal promoter region (CpG sites 18–32 of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA. Conclusion This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune

  9. Development of remote sensing based site specific weed management for Midwest mint production

    Science.gov (United States)

    Gumz, Mary Saumur Paulson

    Peppermint and spearmint are high value essential oil crops in Indiana, Michigan, and Wisconsin. Although the mints are profitable alternatives to corn and soybeans, mint production efficiency must improve in order to allow industry survival against foreign produced oils and synthetic flavorings. Weed control is the major input cost in mint production and tools to increase efficiency are necessary. Remote sensing-based site-specific weed management offers potential for decreasing weed control costs through simplified weed detection and control from accurate site specific weed and herbicide application maps. This research showed the practicability of remote sensing for weed detection in the mints. Research was designed to compare spectral response curves of field grown mint and weeds, and to use these data to develop spectral vegetation indices for automated weed detection. Viability of remote sensing in mint production was established using unsupervised classification, supervised classification, handheld spectroradiometer readings and spectral vegetation indices (SVIs). Unsupervised classification of multispectral images of peppermint production fields generated crop health maps with 92 and 67% accuracy in meadow and row peppermint, respectively. Supervised classification of multispectral images identified weed infestations with 97% and 85% accuracy for meadow and row peppermint, respectively. Supervised classification showed that peppermint was spectrally distinct from weeds, but the accuracy of these measures was dependent on extensive ground referencing which is impractical and too costly for on-farm use. Handheld spectroradiometer measurements of peppermint, spearmint, and several weeds and crop and weed mixtures were taken over three years from greenhouse grown plants, replicated field plots, and production peppermint and spearmint fields. Results showed that mints have greater near infrared (NIR) and lower green reflectance and a steeper red edge slope than

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

    Science.gov (United States)

    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

  11. Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Larsen, Dorthe Helena; Bunkenborg, Jakob;

    2010-01-01

    To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA...... damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia...... telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated...

  12. On the validity of evolutionary models with site-specific parameters.

    Directory of Open Access Journals (Sweden)

    Konrad Scheffler

    Full Text Available Evolutionary models that make use of site-specific parameters have recently been criticized on the grounds that parameter estimates obtained under such models can be unreliable and lack theoretical guarantees of convergence. We present a simulation study providing empirical evidence that a simple version of the models in question does exhibit sensible convergence behavior and that additional taxa, despite not being independent of each other, lead to improved parameter estimates. Although it would be desirable to have theoretical guarantees of this, we argue that such guarantees would not be sufficient to justify the use of these models in practice. Instead, we emphasize the importance of taking the variance of parameter estimates into account rather than blindly trusting point estimates - this is standardly done by using the models to construct statistical hypothesis tests, which are then validated empirically via simulation studies.

  13. Precision farming - Technology assessment of site-specific input application in cereals

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus

    economic and socio-economic analysis. The current status of precision farming in Denmark is as follows: • The technology is primarily applicable for large farm holdings • Economic viability depends on site-specific yield variation • So far, the business economic benefits from most PF-practices are modest...... but it seems possible to obtain a socio-economic benefits from lime, variable rate herbicide and possibly nitrogen application • The technology may improve farm logistics, planning and crop quality (e.g. protein content) - but • The costs of implementing PF-practices are high and • Technical functionality...... several years before the next generation of precision farming systems will be available in practice. Meanwhile, those farmers who already have invested in yield monitors and soil analysis for precision farming should be able to use the current technology in the best possible way....

  14. Polarimetric Kronecker Separability of Site-Specific Double-Directional Channel in an Urban Macrocellular Environment

    Directory of Open Access Journals (Sweden)

    Takada Jun-Ichi

    2009-01-01

    Full Text Available Abstract This paper focuses on the modeling of a double-directional power spectrum density (PSD between the base station (BS and mobile station (MS based on the site-specific measurements in an urban macrocell in Tokyo. First, the authors investigate the Kronecker separability of the joint polarimetric angular PSD between the BS and MS by using the ergodic mutual information. The general form of the sum of channel polarization pair-wise Kronecker product approximation is proposed to be used to model the joint polarimetric angular PSD between the BS and MS. Finally, the double-directional PSD channel model is proposed and verified by comparing the cumulative distribution functions (CDFs of the measured and modeled ergodic mutual information.

  15. Polarimetric Kronecker Separability of Site-Specific Double-Directional Channel in an Urban Macrocellular Environment

    Directory of Open Access Journals (Sweden)

    Kriangsak Sivasondhivat

    2009-01-01

    Full Text Available This paper focuses on the modeling of a double-directional power spectrum density (PSD between the base station (BS and mobile station (MS based on the site-specific measurements in an urban macrocell in Tokyo. First, the authors investigate the Kronecker separability of the joint polarimetric angular PSD between the BS and MS by using the ergodic mutual information. The general form of the sum of channel polarization pair-wise Kronecker product approximation is proposed to be used to model the joint polarimetric angular PSD between the BS and MS. Finally, the double-directional PSD channel model is proposed and verified by comparing the cumulative distribution functions (CDFs of the measured and modeled ergodic mutual information.

  16. Data basis for a site specific radioactive element migration analysis of a repository

    International Nuclear Information System (INIS)

    Migration analysis is of considerable importance in long-term safety analysis of radioactive waste repositories. As a first step the author calculates the transport of radionuclides using data, as far as possible, for an undisturbed hydrogeology. Thereby a reference case is defined. In a later step, possible events and processes can be considered leading to a deviation from the reference case. The present work gives the data base for a selected part of a comprehensive geosphere transport calculation. The report is restricted to a critical evaluation of parameters pertinent to the migration analysis of the 245Cm chain. This includes the important nuclide 237Np. For the first time it is possible to perform a site specific calculation for repositories planned in deep geologic formations in Switzerland. The well known fact that the data basis is extremely sparse is pointed out once more and concretized in detail. (Auth.)

  17. Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface

    Science.gov (United States)

    Faccio, Greta; Kämpf, Michael M.; Piatti, Chiara; Thöny-Meyer, Linda; Richter, Michael

    2014-06-01

    Enzymatic crosslinking of proteins is often limited by the steric availability of the target residues, as of tyrosyl side chains in the case of tyrosinase. Carrying an N-terminal peptide-tag containing two tyrosine residues, the fluorescent protein C-phycocyanin HisCPC from Synechocystis sp. PCC6803 was crosslinked to fluorescent high-molecular weight forms with tyrosinase. Crosslinking with tyrosinase in the presence of L-tyrosine produced non fluorescent high-molecular weight products. Incubated in the presence of tyrosinase, HisCPC could also be immobilized to amino-modified polystyrene beads thus conferring a blue fluorescence. Crosslinking and immobilization were site-specific as both processes required the presence of the N-terminal peptide in HisCPC.

  18. Waste Isolation Pilot Plant (WIPP) five-year Site Specific Plan

    International Nuclear Information System (INIS)

    The Waste Isolation Pilot Plant (WIPP), located 26 miles east of Carlsbad, New Mexico, is a research and development facility vested with the vital mission of demonstrating the safe disposal of radioactive transuranic (TRU) wastes resulting from our nation's defense activities and programs. It is the only facility in the United States specifically designed and constructed for the long-term storage of transuranic wastes. Since the inception of the WIPP project, work has continued to prepare the facility to receive TRU waste. A multitude of studies have been and continue to be conducted to demonstrate the safety of the WIPP facility in accordance with federal and state laws, state agreements, DOE Orders, etc. This Site Specific Plan outlines the implementation of activities at the WIPP project. 12 figs

  19. Efficient Formation of Site-Specific Protein-DNA Hybrids Using Copper-Free Click Chemistry.

    Science.gov (United States)

    Mukhortava, Ann; Schlierf, Michael

    2016-07-20

    Protein-DNA hybrids have become increasingly popular molecular building blocks in bionanotechnology and single-molecule studies to synergistically combine the programmability of DNA with the chemical diversity of proteins. The growing demand for protein-DNA hybrids requires powerful strategies for their conjugation. Here, we present an efficient two-step method for protein-DNA assembly based on copper-free click chemistry. The method allows site-specificity and high coupling efficiency, while maintaining the conservation of protein activity. We compare our method to a commonly used protocol of direct linkage of maleimide-modified oligos. We demonstrate the significantly higher yield with a protein-DNA conjugate, which is analyzed using single-molecule force spectroscopy. PMID:27322198

  20. Site-specific Quantification of Bone Quality using Highly Nonlinear Solitary Waves

    CERN Document Server

    Yang, Jinkyu; Borkowski, Sean; Silvestro, Claudio; De Nardo, Luigi; Daraio, Chiara; Ebramzadeh, Edward

    2010-01-01

    Osteoporosis is a well recognized problem affecting millions of individuals worldwide. Consequently, the need to effectively, efficiently, and affordably diagnose and identify those at risk is essential; moreover, site-specific assessment of bone quality is necessary, not only in the process of risk assessment, but may also be desirable for other applications. The present study evaluated a new one-dimensional granular crystal sensor, composed of a tightly packed chain of beads under Hertzian contact interaction, representing the most suitable fundamental component for solitary wave generation and propagation. First, the sensitivity of the novel sensor was tested using densities of rigid polyurethane foam, representing clinical bone quality ranging from healthy, to severely osteoporotic. Once the relationship between the signal response and known densities was established, the sensor was used to measure several sites located in the proximal femur of ten human cadaveric specimens. The accuracy of the model was ...

  1. A site-specific curated database for the microorganisms of activated sludge and anaerobic digesters

    DEFF Research Database (Denmark)

    McIlroy, Simon Jon; Kirkegaard, Rasmus Hansen; McIlroy, Bianca;

    the composition and dynamics of the most abundant organisms. However, to understand the relationship between the population dynamics and operational parameters of the system, a functional role must be attributed to each organism. The Microbial Database for Activated Sludge (MiDAS) and Anaerobic Digesters (AD......) presented here provides a site specific curated taxonomy for abundant and important microorganisms and integrates it into a community knowledge web platform about the microbes in activated sludge (AS) and their associated ADs (www.midasfieldguide.org). The MiDAS taxonomy, a manual curation of the SILVA...... taxonomy, proposes putative names for each genus-level-taxon that can be used as a common vocabulary for all researchers in the field. The online database covers >250 genera found to be abundant and/or important in biological nutrient removal treatment plants, based on extensive in-house surveys with 16S r...

  2. Drainage filters and constructed wetlands to mitigate site-specific nutrient losses

    DEFF Research Database (Denmark)

    Kjærgaard, Charlotte; Hoffmann, Carl Christian; Iversen, Bo Vangsø;

    nutrient loads from farmland in Denmark. Tile drains and ditches connect fields to receiving waters and act as subsurface highways for both soluble and particulate P and nitrogen. Hence, for a large number of recipients, drainage water nutrient loads has a major impact on water quality, however, mitigation...... options targeting subsurface drainage are lacking. An end-of-pipe drainage filter solution offers the benefits of a targeted measure typically applied to point sources. This calls for a shift of paradigm towards the development of new, cost-efficient technologies to mitigate site-specific nutrient losses...... in drainage. A newly launched Danish research project “SUPREME-TECH” (2010-2015) (www.supreme-tech.dk) funded by the Danish Strategic Research Council, aims at providing the scientific basis for developing cost-effective filter technologies targeting P-retention and N-removal in agricultural subsurface...

  3. Testing the atmospheric dispersion model of CSA N288.1 with site-specific data

    CERN Document Server

    Chouhan, S L

    2001-01-01

    The atmospheric dispersion component of CSA Standard N288. 1, which provides guidelines for calculating derived release limits, has been tested. Long-term average concentrations of tritium in air were predicted using site-specific release rates and meteorological data and compared with measured concentrations at 43 monitoring sites at all CANDU stations in Canada. The predictions correlate well with the observations but were found to be conservative, overestimating by about 50% on average. The model overpredicted 84% of the time, with the highest prediction lying a factor of 5.5 above the corresponding observation. The model underpredicted the remaining 16% of the time, with the lowest prediction about one-half of the corresponding measurement. Possible explanations for this bias are discussed but no single reason appears capable of accounting for the discrepancy. Rather, the tendency to overprediction seems to result from the cumulative effects of a number of small conservatisms in the model. The model predi...

  4. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    Science.gov (United States)

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  5. Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions

    Directory of Open Access Journals (Sweden)

    Mukherjee Srabani

    2003-01-01

    Full Text Available During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP. Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches.

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

    Science.gov (United States)

    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

  7. Site-specific time heterogeneity of the substitution process and its impact on phylogenetic inference

    Directory of Open Access Journals (Sweden)

    Philippe Hervé

    2011-01-01

    Full Text Available Abstract Background Model violations constitute the major limitation in inferring accurate phylogenies. Characterizing properties of the data that are not being correctly handled by current models is therefore of prime importance. One of the properties of protein evolution is the variation of the relative rate of substitutions across sites and over time, the latter is the phenomenon called heterotachy. Its effect on phylogenetic inference has recently obtained considerable attention, which led to the development of new models of sequence evolution. However, thus far focus has been on the quantitative heterogeneity of the evolutionary process, thereby overlooking more qualitative variations. Results We studied the importance of variation of the site-specific amino-acid substitution process over time and its possible impact on phylogenetic inference. We used the CAT model to define an infinite mixture of substitution processes characterized by equilibrium frequencies over the twenty amino acids, a useful proxy for qualitatively estimating the evolutionary process. Using two large datasets, we show that qualitative changes in site-specific substitution properties over time occurred significantly. To test whether this unaccounted qualitative variation can lead to an erroneous phylogenetic tree, we analyzed a concatenation of mitochondrial proteins in which Cnidaria and Porifera were erroneously grouped. The progressive removal of the sites with the most heterogeneous CAT profiles across clades led to the recovery of the monophyly of Eumetazoa (Cnidaria+Bilateria, suggesting that this heterogeneity can negatively influence phylogenetic inference. Conclusion The time-heterogeneity of the amino-acid replacement process is therefore an important evolutionary aspect that should be incorporated in future models of sequence change.

  8. phiC31 integrase-mediated site-specific recombination in barley.

    Directory of Open Access Journals (Sweden)

    Eszter Kapusi

    Full Text Available 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 F(1, 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.

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

    Science.gov (United States)

    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.

  10. Site-Specific Seismic Site Response Model for the Waste Treatment Plant, Hanford, Washington

    International Nuclear Information System (INIS)

    The seismic design basis for the Waste Treatment Plant (WTP) at the Department of Energy's Hanford Site near Richland, Washington, was established in 1999 based on an extensive probabilistic seismic hazard analysis completed in 1996 by Geomatrix Consultants, Inc. In subsequent years, the Defense Nuclear Facilities Safety Board (DNFSB) staff questioned the some of the assumptions used in developing the seismic design basis, particularly the adequacy of the site geotechnical surveys. Existing site-specific shear wave velocity data were considered insufficient to reliably use California earthquake response data to directly predict ground motions at the Hanford Site. To address this concern, the Department of Energy's Office of River Protection (ORP) and Pacific Northwest National Laboratory (PNNL) developed and executed a plan for acquiring site-specific soil data down to approximately 500 feet, and for reanalyzing the effects of deeper layers of sediments interbedded with basalt. New geophysical data were acquired, analyzed, and interpreted with respect to existing geologic information gathered from other Hanford-related projects in the WTP area. Existing data from deep boreholes were assembled and interpreted to produce a model of the deeper rock layers consisting of inter-layered basalts and sedimentary interbeds. These data were analyzed statistically to determine the variability of seismic velocities. The earthquake ground motion response was simulated on a large number of models resulting from a weighted logic tree approach that addressed the geologic and geophysical uncertainties. Weights in the logic tree were chosen by a working group based on the strength or weakness of the available data for each combination of logic tree parameters. Finally, interim design ground motion spectra were developed to envelope the remaining uncertainties. The results of this study demonstrate that the site-specific soil structure (Hanford and Ringold formations) beneath the WTP

  11. PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities

    Institute of Scientific and Technical Information of China (English)

    Yun-xia YANG; Yun FENG; Bo-yao WANG; Qi WU

    2004-01-01

    AIM: To construct PGEX-1λT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA- 1 cell mRNA and the prokaryotic expression vector PGEX- 1λT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells. RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseltdomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO2-4 containing Medium E. CONCLUSION: PCR-based mutagensis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping α-helical conformation of FALL-39 and increasing net positive charge can increase the

  12. Environmental Management Waste Management Facility (EMWMF) Site-Specific Health and Safety Plan, Oak Ridge, Tennessee

    Energy Technology Data Exchange (ETDEWEB)

    Flynn, N.C. Bechtel Jacobs

    2008-04-21

    The Bechtel Jacobs Company LLC (BJC) policy is to provide a safe and healthy workplace for all employees and subcontractors. The implementation of this policy requires that operations of the Environmental Management Waste Management Facility (EMWMF), located one-half mile west of the U.S. Department of Energy (DOE) Y-12 National Security Complex, be guided by an overall plan and consistent proactive approach to environment, safety and health (ES&H) issues. The BJC governing document for worker safety and health, BJC/OR-1745, 'Worker Safety and Health Program', describes the key elements of the BJC Safety and Industrial Hygiene (IH) programs, which includes the requirement for development and implementation of a site-specific Health and Safety Plan (HASP) where required by regulation (refer also to BJC-EH-1012, 'Development and Approval of Safety and Health Plans'). BJC/OR-1745, 'Worker Safety and Health Program', implements the requirements for worker protection contained in Title 10 Code of Federal Regulations (CFR) Part 851. The EMWMF site-specific HASP requirements identifies safe operating procedures, work controls, personal protective equipment, roles and responsibilities, potential site hazards and control measures, site access requirements, frequency and types of monitoring, site work areas, decontamination procedures, and outlines emergency response actions. This HASP will be available on site for use by all workers, management and supervisors, oversight personnel and visitors. All EMWMF assigned personnel will be briefed on the contents of this HASP and will be required to follow the procedures and protocols as specified. The policies and procedures referenced in this HASP apply to all EMWMF operations activities. In addition the HASP establishes ES&H criteria for the day-to-day activities to prevent or minimize any adverse effect on the environment and personnel safety and health and to meet standards that define acceptable

  13. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Yuan,Y.; Pei, Y.; Ma, J.; Kuryavyi, V.; Zhadina, M.; Meister, G.; Chen, H.; Dauter, Z.; Tuschi, T.; Patel, D.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

  14. Exploring the effects of linker composition on site-specifically modified antibody-drug conjugates.

    Science.gov (United States)

    Albers, Aaron E; Garofalo, Albert W; Drake, Penelope M; Kudirka, Romas; de Hart, Gregory W; Barfield, Robyn M; Baker, Jeanne; Banas, Stefanie; Rabuka, David

    2014-12-17

    In the context of antibody-drug conjugates (ADCs), noncleavable linkers provide a means to deliver cytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted release of the free drug. Additionally, noncleavable linkers afford an opportunity to change the chemical properties of the small molecule to improve potency or diminish affinity for multidrug transporters, thereby improving efficacy. We employed the aldehyde tag coupled with the hydrazino-iso-Pictet-Spengler (HIPS) ligation to generate a panel of site-specifically conjugated ADCs that varied only in the noncleavable linker portion. The ADC panel comprised antibodies carrying a maytansine payload ligated through one of five different linkers. Both the linker-maytansine constructs alone and the resulting ADC panel were characterized in a variety of in vitro and in vivo assays measuring biophysical and functional properties. We observed that slight differences in linker design affected these parameters in disparate ways, and noted that efficacy could be improved by selecting for particular attributes. These studies serve as a starting point for the exploration of more potent noncleavable linker systems. PMID:25176286

  15. Site specific monitoring of multiple information systems - the HappyFace Project

    Science.gov (United States)

    Büge, Volker; Mauch, Viktor; Quast, Günter; Scheurer, Armin; Trunov, Artem

    2010-04-01

    An efficient administration of computing centres requires sophisticated tools for the monitoring of the local infrastructure. Sharing such resources in a grid infrastructure, like the Worldwide LHC Computing Grid (WLCG), goes ahead with a large number of external monitoring systems, offering information on the status of the services and user jobs at a grid site. This huge flood of information from many different sources retards the identification of problems and complicates the local administration. In addition, the web interfaces for the access to the site specific information are often very slow and uncomfortable to use. A meta-monitoring system which automatically queries the different relevant monitoring systems could provide a fast and comfortable access to all important information for the local administration. It becomes also feasible to easily correlate information from different sources and provides an easy access also for non-expert users. In this paper, we describe the HappyFace Project, a modular software framework for such purpose. It queries existing monitoring sources and processes the results to provide a single point of entrance for information on a grid site and its specific services.

  16. Assembled modules technology for site-specific prolonged delivery of norfloxacin.

    Science.gov (United States)

    Oliveira, Paulo Renato; Bernardi, Larissa Sakis; Strusi, Orazio Luca; Mercuri, Salvatore; Segatto Silva, Marcos A; Colombo, Paolo; Sonvico, Fabio

    2011-02-28

    The aim of this research was to design and study norfloxacin (NFX) release in floating conditions from compressed hydrophilic matrices of hydroxypropylmethylcellulose (HPMC) or poly(ethylene oxide) (PEO). Module assembling technology for drug delivery system manufacturing was used. Two differently cylindrical base curved matrix/modules, identified as female and male, were assembled in void configuration by friction interlocking their concave bases obtaining a floating release system. Drug release and floatation behavior of this assembly was investigated. Due to the higher surface area exposed to the release medium, faster release was observed for individual modules compared to their assembled configuration, independently on the polymer used and concentration. The release curves analyzed using the Korsmeyer exponential equation and Peppas & Sahlin binomial equation showed that the drug release was controlled both by drug diffusion and polymer relaxation or erosion mechanisms. However, convective transport was predominant with PEO and at low content of polymers. NFX release from PEO polymeric matrix was more erosion dependent than HPMC. The assembled systems were able to float in vitro for up to 240min, indicating that this drug delivery system of norfloxacin could provide gastro-retentive site-specific release for increasing norfloxacin bioavailability. PMID:21134427

  17. Gated rotation mechanism of site-specific recombination by ϕC31 integrase.

    Science.gov (United States)

    Olorunniji, Femi J; Buck, Dorothy E; Colloms, Sean D; McEwan, Andrew R; Smith, Margaret C M; Stark, W Marshall; Rosser, Susan J

    2012-11-27

    Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

  18. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective.

    Science.gov (United States)

    Jayaram, Makkuni; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Guga, Piotr; Fan, Hsui-Fang; Voziyanov, Yuri

    2015-08-01

    Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been employed to trace the topological features of DNA within high-order DNA interactions established by protein machines. The directed evolution of altered specificity YRs, combined with their spatially and temporally regulated expression, heralds their emergence as vital tools in genome engineering projects with wide-ranging biotechnological and medical applications. PMID:26350308

  19. Site-Specific Analysis of Geothermal Development-Data Files of Prospective Sites

    Energy Technology Data Exchange (ETDEWEB)

    Williams, F.; Cohen, A.; Pfundstein, R.; Pond, S.

    1977-10-01

    This document presents site-specific data and sample development schedules for the first plant on line at 30 hydrothermal and 7 geopressured prospective sites (prospects) that are believed to be suited for supporting the geothermal generation of electricity. This report includes many prospects from an earlier METREK report on geothermal development scenarios. The list has been augmented with other sites chosen as development prospects by the division of Geothermal Energy (DGE) of the Energy Research and Development Administration (ERDA). The DGE additions include a general area called ''Cascade Range''. METREK has chosen the following specific Cascade Range Sites in place of that general area: Baker Hot Springs, Mount Hood, Lassen and Glass Mountain/ Diablo. All the prospects have been selected on the basis of current knowledge of hydrothermal and geopressured resources. The selection is intended for program planning purposes. Neither METREK nor the Federal government warrants that any of these sites will necessarily be developed, nor does their selection necessarily imply any commitment on the part of the Federal government to their development. [DJE-2005

  20. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

    Science.gov (United States)

    Onouchi, H; Yokoi, K; Machida, C; Matsuzaki, H; Oshima, Y; Matsuoka, K; Nakamura, K; Machida, Y

    1991-12-11

    Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

  1. Relationship of Height to Site-Specific Fracture Risk in Postmenopausal Women.

    Science.gov (United States)

    Armstrong, Miranda Eg; Kirichek, Oksana; Cairns, Benjamin J; Green, Jane; Reeves, Gillian K

    2016-04-01

    Height has been associated with increased risk of fracture of the neck of femur. However, information on the association of height with fractures at other sites is limited and conflicting. A total of 796,081 postmenopausal women, who reported on health and lifestyle factors including a history of previous fractures and osteoporosis, were followed for 8 years for incident fracture at various sites by record linkage to National Health Service hospital admission data. Adjusted relative risks of fracture at different sites per 10-cm increase in height were estimated using Cox regression. Numbers with site-specific fractures were: humerus (3036 cases), radius and/or ulna (1775), wrist (9684), neck of femur (5734), femur (not neck) (713), patella (649), tibia and/or fibula (1811), ankle (5523), and clavicle/spine/rib (2174). The risk of fracture of the neck of femur increased with increasing height (relative risk [RR] = 1.48 per 10-cm increase, 99% confidence interval [CI] 1.39-1.57) and the proportional increase in risk was significantly greater than for all other fracture sites (pheterogeneity < 0.001). For the other sites, fracture risk also increased with height (RR = 1.15 per 10 cm, CI 1.12-1.18), but there was only very weak evidence of a possible difference in risk between the sites (pheterogeneity = 0.03). In conclusion, taller women are at increased risk of fracture, especially of the neck of femur. PMID:26572496

  2. Site-specific waste management instruction for the Onsite Measurements Services Organization

    International Nuclear Information System (INIS)

    This Site-Specific Waste Management Instruction provides guidance for the management of waste generated as a result of field screening measurements performed by the Onsite Measurements Services Organization. Because this report is not project specific, it is not possible to designate waste streams through this document, other than to assess whether the analyses themselves will cause a waste stream to be designated. Generally, field screening methodologies that do not create dangerous waste are employed instead of those that create dangerous waste. The analyses within the scope of this SSWMI are as follows: VOC analyses of water samples using aqueous headspace analyses; VOC analyses of air samples using soil gas analytical techniques; total uranium analyses of water samples using kinetic phosphorescence analyses; chromate analyses of water samples is using a colorimetric method based on ''AccuVac'' ampuls manufactured by the Hach Company; nitrate analyses of water samples using paper test strips; conductivity analyses of water samples using electrodes; and sulfate analyses of water samples using a colorimetric method based on ''AccuVac'' ampuls

  3. MUCOADHESIVE MICROPARTICLES OF CARBOXYMETHYL CHITOSAN FOR SITE SPECIFIC DELIVERY OF PANTOPRAZOLE: FORMULATION AND IN VITRO CHARACTERIZATION

    Directory of Open Access Journals (Sweden)

    N.A. Gujarathi*, B.R. Rane and J.K. Patel

    2012-11-01

    Full Text Available Carboxymethyl chitosan, a water soluble modified carboxymethyl substituted chitosan derivative have distinct and unique properties, rendering them effective to form selective permeable mucoadhesive film or membranes. In the formulation of chitosan microsphere an acidic environment is essentially required that may degrade acid sensitive moiety, peptide or protein drugs. Mucoadhesive microparticle of carboxymethyl chitosan was designed and developed for site specific sustained release of Pantoprazole sodium. Thus prolong the residence time at the absorption site by intimate contact with the mucus layer thereby increase bioavailability, reduce the frequency of dose administration and also prolong the drug release. The mucoadhesive microparticles were prepared by Orifice ionic gelation method using carboxymethyl chitosan in combination with Carbopol 934 and HPMC K15. Entrapment efficiency was in the range of 42.4 to 84.6 %. SEM studies revealed that microparticles were discrete, spherical and free flowing. Microparticle exhibited good mucoadhesive property in the in vitro wash off test and found that Carbopol 934 had greater mucoadhesive strength than that of HPMC K15. A sustained release of Pantoprazole sodium was obtained from mucoadhesive microparticle. Stability study of optimized batch was carried out and drug content found was retained with permissible limits and there was no significant difference in the drug content.

  4. Amorphous Semiconductor Nanowires Created by Site-Specific Heteroatom Substitution with Significantly Enhanced Photoelectrochemical Performance.

    Science.gov (United States)

    He, Ting; Zu, Lianhai; Zhang, Yan; Mao, Chengliang; Xu, Xiaoxiang; Yang, Jinhu; Yang, Shihe

    2016-08-23

    Semiconductor nanowires that have been extensively studied are typically in a crystalline phase. Much less studied are amorphous semiconductor nanowires due to the difficulty for their synthesis, despite a set of characteristics desirable for photoelectric devices, such as higher surface area, higher surface activity, and higher light harvesting. In this work of combined experiment and computation, taking Zn2GeO4 (ZGO) as an example, we propose a site-specific heteroatom substitution strategy through a solution-phase ions-alternative-deposition route to prepare amorphous/crystalline Si-incorporated ZGO nanowires with tunable band structures. The substitution of Si atoms for the Zn or Ge atoms distorts the bonding network to a different extent, leading to the formation of amorphous Zn1.7Si0.3GeO4 (ZSGO) or crystalline Zn2(GeO4)0.88(SiO4)0.12 (ZGSO) nanowires, respectively, with different bandgaps. The amorphous ZSGO nanowire arrays exhibit significantly enhanced performance in photoelectrochemical water splitting, such as higher and more stable photocurrent, and faster photoresponse and recovery, relative to crystalline ZGSO and ZGO nanowires in this work, as well as ZGO photocatalysts reported previously. The remarkable performance highlights the advantages of the ZSGO amorphous nanowires for photoelectric devices, such as higher light harvesting capability, faster charge separation, lower charge recombination, and higher surface catalytic activity. PMID:27494205

  5. Development of Floating-Mucoadhesive Microsphere for Site Specific Release of Metronidazole

    Science.gov (United States)

    Amin, Md. Lutful; Ahmed, Tajnin; Mannan, Md. Abdul

    2016-01-01

    Purpose: The purpose of this study was to develop and evaluate metronidazole loaded floating-mucoadhesive microsphere for sustained drug release at the gastric mucosa. Methods: Alginate gastroretentive microspheres containing metronidazole were prepared by ionic gelation method using sodium bicarbonate as gas forming agent, guar gum as mucoadhesive polymer, and Eudragit L100 as drug release modifier. Carbopol was used for increasing the bead strength. The microspheres were characterized by scanning electron microscopy and evaluated by means of drug entrapment efficiency, in vitro buoyancy, and swelling studies. In vitro mucoadhesion and drug release studies were carried out in order to evaluate site specific sustained drug release. Results: All formulations showed 100% buoyancy in vitro for a prolonged period of time. Amount of guar gum influenced the properties of different formulations. The formulation containing drug and guar gum at a ratio of 1:0.5 showed the best results with 76.3% drug entrapment efficiency, 61.21% mucoadhesion, and sustained drug release. Carbopol was found to increase surface smoothness of the microspheres. Conclusion: Metronidazole mucoadhesive-floating microspheres can be effectively used for sustained drug release to the gastric mucosa in treatment of upper GIT infection. PMID:27478781

  6. Development of a novel DDS for site-specific PEGylated proteins

    Directory of Open Access Journals (Sweden)

    Yoshioka Yasuo

    2011-05-01

    Full Text Available Abstract Because of the shifted focus in life science research from genome analyses to genetic and protein function analyses, we now know functions of numerous proteins. These analyses, including those of newly identified proteins, are expected to contribute to the identification of proteins of therapeutic value in various diseases. Consequently, pharmacoproteomic-based drug discovery and development of protein therapies attracted a great deal of attention in recent years. Clinical applications of most of these proteins are, however, limited because of their unexpectedly low therapeutic effects, resulting from the proteolytic degradation in vivo followed by rapid removal from the circulatory system. Therefore, frequent administration of excessively high dose of a protein is required to observe its therapeutic effect in vivo. This often results in impaired homeostasis in vivo and leads to severe adverse effects. To overcome these problems, we have devised a method for chemical modification of proteins with polyethylene glycol (PEGylation and other water-soluble polymers. In addition, we have established a method for creating functional mutant proteins (muteins with desired properties, and developed a site-specific polymer-conjugation method to further improve their therapeutic potency. In this review, we are introducing our original protein-drug innovation system mentioned above.

  7. Design of a Soil Cutting Resistance Sensor for Application in Site-Specific Tillage

    Directory of Open Access Journals (Sweden)

    Manuel Perez-Ruiz

    2013-05-01

    Full Text Available One objective of precision agriculture is to provide accurate information about soil and crop properties to optimize the management of agricultural inputs to meet site-specific needs. This paper describes the development of a sensor equipped with RTK-GPS technology that continuously and efficiently measures soil cutting resistance at various depths while traversing the field. Laboratory and preliminary field tests verified the accuracy of this prototype soil strength sensor. The data obtained using a hand-operated soil cone penetrometer was used to evaluate this field soil compaction depth profile sensor. To date, this sensor has only been tested in one field under one gravimetric water content condition. This field test revealed that the relationships between the soil strength profile sensor (SSPS cutting force and soil cone index values are assumed to be quadratic for the various depths considered: 0–10, 10–20 and 20–30 cm (r2 = 0.58, 0.45 and 0.54, respectively. Soil resistance contour maps illustrated its practical value. The developed sensor provides accurate, timely and affordable information on soil properties to optimize resources and improve agricultural economy.

  8. Hanford Integrated Planning Process: 1993 Hanford Site-specific science and technology plan

    Energy Technology Data Exchange (ETDEWEB)

    1993-12-01

    This document is the FY 1993 report on Hanford Site-specific science and technology (S&T) needs for cleanup of the Site as developed via the Hanford Integrated Planning Process (HIPP). It identifies cleanup problems that lack demonstrated technology solutions and technologies that require additional development. Recommendations are provided regarding allocation of funding to address Hanford`s highest-priority technology improvement needs, technology development needs, and scientific research needs, all compiled from a Sitewide perspective. In the past, the S&T agenda for Hanford Site cleanup was sometimes driven by scientists and technologists, with minimal input from the ``problem owners`` (i.e., Westinghouse Hanford Company [WHC] staff who are responsible for cleanup activities). At other times, the problem-owners made decisions to proceed with cleanup without adequate scientific and technological inputs. Under both of these scenarios, there was no significant stakeholder involvement in the decision-making process. One of the key objectives of HIPP is to develop an understanding of the integrated S&T requirements to support the cleanup mission, (a) as defined by the needs of the problem owners, the values of the stakeholders, and the technology development expertise that exists at Hanford and elsewhere. This requires a periodic, systematic assessment of these needs and values to appropriately define a comprehensive technology development program and a complementary scientific research program. Basic to our success is a methodology that is defensible from a technical perspective and acceptable to the stakeholders.

  9. Simulation-Based Approach for Site-Specific Optimization of Hydrokinetic Turbine Arrays

    Science.gov (United States)

    Sotiropoulos, F.; Chawdhary, S.; Yang, X.; Khosronejad, A.; Angelidis, D.

    2014-12-01

    A simulation-based approach has been developed to enable site-specific optimization of tidal and current turbine arrays in real-life waterways. The computational code is based on the St. Anthony Falls Laboratory Virtual StreamLab (VSL3D), which is able to carry out high-fidelity simulations of turbulent flow and sediment transport processes in rivers and streams taking into account the arbitrary geometrical complexity characterizing natural waterways. The computational framework can be used either in turbine-resolving mode, to take into account all geometrical details of the turbine, or with the turbines parameterized as actuator disks or actuator lines. Locally refined grids are employed to dramatically increase the resolution of the simulation and enable efficient simulations of multi-turbine arrays. Turbine/sediment interactions are simulated using the coupled hydro-morphodynamic module of VSL3D. The predictive capabilities of the resulting computational framework will be demonstrated by applying it to simulate turbulent flow past a tri-frame configuration of hydrokinetic turbines in a rigid-bed turbulent open channel flow as well as turbines mounted on mobile bed open channels to investigate turbine/sediment interactions. The utility of the simulation-based approach for guiding the optimal development of turbine arrays in real-life waterways will also be discussed and demonstrated. This work was supported by NSF grant IIP-1318201. Simulations were carried out at the Minnesota Supercomputing Institute.

  10. Five guidelines for the evaluation of site-specific medium range probabilistic temperature forecasts

    CERN Document Server

    Jewson, S; Jewson, Stephen; Ziehmann, Christine

    2003-01-01

    Probabilistic temperature forecasts are potentially useful to the energy and weather derivatives industries. However, at present, they are little used. There are a number of reasons for this, but we believe this is in part due to inadequacies in the methodologies that have been used to evaluate such forecasts, leading to uncertainty as to whether the forecasts are really useful or not and making it hard to work out which forecasts are best. To remedy this situation we describe a set of guidelines that we recommend should be followed when evaluating the skill of site-specific probabilistic medium range temperature forecasts. If these guidelines are followed then the results of validation can be used directly by forecast users to make decisions about which forecasts to use. If they are not followed then the results of validation may be interesting, but will not be practically useful for users. We find that none of the published studies that evaluate such forecasts fall within our guidelines, and that, as a resu...

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

    Science.gov (United States)

    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.

  12. Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination.

    Science.gov (United States)

    Buchholz, F; Ringrose, L; Angrand, P O; Rossi, F; Stewart, A F

    1996-11-01

    Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems. To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays. In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre. FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C. Cre is optimally efficient at 37 degrees C and above. Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures. We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile. The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage. Our results recommend the use of Cre for applications in mice that require efficient recombination. The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.

  13. Site-specific probing of charge transfer dynamics in organic photovoltaics

    Energy Technology Data Exchange (ETDEWEB)

    Arion, Tiberiu; Roth, Friedrich [Center for Free-Electron Laser Science/DESY, Notkestraße 85, D-22607 Hamburg (Germany); Neppl, Stefan; Shavorskiy, Andrey; Bluhm, Hendrik; Gessner, Oliver [Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (United States); Hussain, Zahid [ALS, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (United States); Eberhardt, Wolfgang [Center for Free-Electron Laser Science/DESY, Notkestraße 85, D-22607 Hamburg (Germany); ALS, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (United States); Institute of Optics and Atomic Physics, TU Berlin, Straße des 17. Juni 135, D-10623 Berlin (Germany)

    2015-03-23

    We report the site-specific probing of charge-transfer dynamics in a prototype system for organic photovoltaics (OPVs) by picosecond time-resolved X-ray photoelectron spectroscopy. A layered system consisting of approximately two monolayers of C{sub 60} deposited on top of a thin film of Copper-Phthalocyanine (CuPC) is excited by an optical pump pulse and the induced electronic dynamics are probed with 590 eV X-ray pulses. Charge transfer from the electron donor (CuPC) to the acceptor (C{sub 60}) and subsequent charge carrier dynamics are monitored by recording the time-dependent C 1s core level photoemission spectrum of the system. The arrival of electrons in the C{sub 60} layer is readily observed as a completely reversible, transient shift of the C{sub 60} associated C 1s core level, while the C 1s level of the CuPC remains unchanged. The capability to probe charge transfer and recombination dynamics in OPV assemblies directly in the time domain and from the perspective of well-defined domains is expected to open additional pathways to better understand and optimize the performance of this emerging technology.

  14. Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.

    Directory of Open Access Journals (Sweden)

    Munehiro Uemura

    Full Text Available Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells. Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.

  15. A Site-Specific Index Based on Weathering Forms Visible in Central Oxford, UK

    Directory of Open Access Journals (Sweden)

    Mary J. Thornbush

    2012-11-01

    Full Text Available The authenticity of much of the stone-work along Queen’s Lane in central Oxford, UK presented an opportunity to produce a photographic survey from which a weathering index could be established. This represents a site-specific approach to devising a weathering form. Because it is photo-based, weathering forms are visible for comparison and classification purposes across disciplines. Limestone pertaining to building ashlar and plinths along this roadway, which mainly belong to Queen’s College, St Edmund Hall, New College, and Hertford College, was classified according to this newly introduced weathering index, the size-extent (S-E index, through consideration of type, size, extent, impact, and trigger. This size- (range and extent-based classification system enables for the assessment of weathering forms of various types, including soiling and decay features as well as those potentially expected in the presence of vegetation and animals. Weathering forms of a range of sizes were present, with a slightly greater abundance of small types (mm-cm in the micro- to mesoscale and more discrete types with a low extent. For this location in central Oxford, chemical weathering was found to be the predominant type of soiling and decay.

  16. Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site-Specifically Modified with Argpyrimidine.

    Science.gov (United States)

    Matveenko, Maria; Cichero, Elena; Fossa, Paola; Becker, Christian F W

    2016-09-12

    Non-enzymatic posttranslational modifications (nPTMs) affect at least ∼30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site-specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site-selective protein modification. PMID:27440458

  17. Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System.

    Science.gov (United States)

    Mettrick, Karla A; Lawrence, Nikki; Mason, Claire; Weaver, Georgia M; Corocher, Tayla-Ann; Grainge, Ian

    2016-01-01

    Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system. PMID:27583408

  18. Site-Specific Variability in the Chemical Diversity of the Antarctic Red Alga Plocamium cartilagineum

    Directory of Open Access Journals (Sweden)

    Ryan M. Young

    2013-06-01

    Full Text Available Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like “P. cartilagineum” from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km2 area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.

  19. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    Science.gov (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  20. Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

    Science.gov (United States)

    Bhattacharjee, Somnath; Liu, Wenge; Wang, Wei-Han; Weitzhandler, Isaac; Li, Xinghai; Qi, Yizhi; Liu, Jinyao; Pang, Yan; Hunt, Donald F; Chilkoti, Ashutosh

    2015-11-01

    Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

  1. Hanford Integrated Planning Process: 1993 Hanford Site-specific science and technology plan

    International Nuclear Information System (INIS)

    This document is the FY 1993 report on Hanford Site-specific science and technology (S ampersand T) needs for cleanup of the Site as developed via the Hanford Integrated Planning Process (HIPP). It identifies cleanup problems that lack demonstrated technology solutions and technologies that require additional development. Recommendations are provided regarding allocation of funding to address Hanford's highest-priority technology improvement needs, technology development needs, and scientific research needs, all compiled from a Sitewide perspective. In the past, the S ampersand T agenda for Hanford Site cleanup was sometimes driven by scientists and technologists, with minimal input from the ''problem owners'' (i.e., Westinghouse Hanford Company [WHC] staff who are responsible for cleanup activities). At other times, the problem-owners made decisions to proceed with cleanup without adequate scientific and technological inputs. Under both of these scenarios, there was no significant stakeholder involvement in the decision-making process. One of the key objectives of HIPP is to develop an understanding of the integrated S ampersand T requirements to support the cleanup mission, (a) as defined by the needs of the problem owners, the values of the stakeholders, and the technology development expertise that exists at Hanford and elsewhere. This requires a periodic, systematic assessment of these needs and values to appropriately define a comprehensive technology development program and a complementary scientific research program. Basic to our success is a methodology that is defensible from a technical perspective and acceptable to the stakeholders

  2. Site specific probability of passive acoustic detection of humpback whale calls from single fixed hydrophones.

    Science.gov (United States)

    Helble, Tyler A; D'Spain, Gerald L; Hildebrand, John A; Campbell, Gregory S; Campbell, Richard L; Heaney, Kevin D

    2013-09-01

    Passive acoustic monitoring of marine mammal calls is an increasingly important method for assessing population numbers, distribution, and behavior. A common mistake in the analysis of marine mammal acoustic data is formulating conclusions about these animals without first understanding how environmental properties such as bathymetry, sediment properties, water column sound speed, and ocean acoustic noise influence the detection and character of vocalizations in the acoustic data. The approach in this paper is to use Monte Carlo simulations with a full wave field acoustic propagation model to characterize the site specific probability of detection of six types of humpback whale calls at three passive acoustic monitoring locations off the California coast. Results show that the probability of detection can vary by factors greater than ten when comparing detections across locations, or comparing detections at the same location over time, due to environmental effects. Effects of uncertainties in the inputs to the propagation model are also quantified, and the model accuracy is assessed by comparing calling statistics amassed from 24,690 humpback units recorded in the month of October 2008. Under certain conditions, the probability of detection can be estimated with uncertainties sufficiently small to allow for accurate density estimates. PMID:23968053

  3. Site-specific waste management instruction for the field sampling organization

    International Nuclear Information System (INIS)

    The Site-Specific Waste Management Instruction (SSWMI) provides guidance for the management of waste generated from field-sampling activities performed by the Environment Restoration Contractor (ERC) Sampling Organization that are not managed as part of a project SSWMI. Generally, the waste is unused preserved groundwater trip blanks, used and expired calibration solutions, and other similar waste that cannot be returned to an ERC project for disposal. The specific waste streams addressed by this SSWMI are identified in Section 2.0. This SSWMI was prepared in accordance with BHI-EE-02, Environmental Requirements. Waste generated from field sample collection activities should be returned to the project and managed in accordance with the applicable project-specific SSWMI whenever possible. However, returning all field sample collection and associated waste to a project for disposal may not always be practical or cost effective. Therefore, the ERC field sampling organization must manage and arrange to dispose of the waste using the (Bechtel Hanford, Inc. [BHI]) Field Support Waste Management (FSWM) services. This SSWMI addresses those waste streams that are the responsibility of the field sampling organization to manage and make arrangements for disposal

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

    Science.gov (United States)

    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. Site-specific, adult bone benefits attributed to loading during youth: A preliminary longitudinal analysis.

    Science.gov (United States)

    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 (pGYM BMC (pGYM endosteal diameter (pGYM advantages in narrow neck cortical thickness and buckling ratio (both pGYM than NON (pGYM 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 observation is necessary to determine whether residual loading benefits enhance lifelong skeletal strength.

  6. Rapid kinematic slip inversion with regional geophysical data: towards site-specific tsunami intensity forecasts.

    Science.gov (United States)

    Melgar, D.; Bock, Y.

    2014-12-01

    Rapid kinematic slip inversions immediately following earthquake rupture is traditionally limited to teleseismic data and delayed many hours after large events. Regional data such as strong motion is difficult to incorporate quickly into images of the source process because baseline offsets render the long period portion of the recording unreliable. Recently it's been demonstrated that high rate GPS can potentially produce rapid slip inversions for large events but is limited to very long periods. With an example of the 2011 M9 Tohoku-oki event we will demonstrate that the optimal on-the-fly combination of GPS and strong motion through a seismogeodetic Kalman filter produces reliable, broadband strong motion displacement and velocity waveforms that can be used for kinematic inversion. Through joint inversion of displacement and velocity waveforms we will show that it is possible to obtain a broadband image of the source. Furthermore, we will also show that it is possible to include offshore geophysical observables such as sea surface measurements of tsunami propagation from GPS buoys and ocean bottom pressure sensors into the kinematic inversion. These data better constrain the shallowest part of rupture. We will use the time-dependent deformation of bathymetry predicted from the inversion results as an initial condition for tsunami propagation and inundation modeling. Through a comparison to post-event survey observations we will demonstrate that it is possible to reproduce the inundation pattern along the coastline in great detail and argue that detailed site-specific forecast of tsunami intensity is achievable with current methods and instrumentation.

  7. Current implementation of site-specific technologies in U.S. cotton production

    Science.gov (United States)

    Barnes, Edward M.

    2004-11-01

    The initial adoption of site-specific management for cotton production was slower than by commodities grown in the mid-west. Part of the delayed adoption can be explained by the lack of functional cotton yield monitors. Now that yield monitors are commercially available, cotton producers are beginning to find many applications for geospatial technologies. The emphasis of this paper is on applications that are being implemented at some level on commercial farms. Traditional grid-based soil sampling has found some use for pre-plant application of fertilizer, and soil conductivity mapping has been used to apply variable rate soil amendments in the west. During the growing season, vegetation indices such as the normalized difference vegetation index (NDVI) have been used as a tool to direct scouting for insect infestations. The scouting information and NDVI are combined to create variable rate insecticide application maps. A catalyst to this approach has been the recent development of aerial variable rate application technology. In some production regions, it is necessary to control cotton's vegetative development rate with plant growth regulators (PGRs). Vegetation indices are very useful for defining areas of the field where PGRs are needed. Research is also being conducted on the use of imagery for the development of defoliation application maps before harvest. In the short-term, simple vegetation indices can meet many of management information needs for cotton when combined with directed scouting of the field. However, to be ultimately successful, dependable and frequent sources of imagery will be required. Near-real time delivery is essential, as a majority of the management decisions are often made on a daily basis. Most producer-based applications of these data have been from airborne platforms managed by local image providers where flexible image acquisition schedules are possible.

  8. Configuration and specifications of an Unmanned Aerial Vehicle (UAV) for early site specific weed management.

    Science.gov (United States)

    Torres-Sánchez, Jorge; López-Granados, Francisca; De Castro, Ana Isabel; Peña-Barragán, José Manuel

    2013-01-01

    A new aerial platform has risen recently for image acquisition, the Unmanned Aerial Vehicle (UAV). This article describes the technical specifications and configuration of a UAV used to capture remote images for early season site- specific weed management (ESSWM). Image spatial and spectral properties required for weed seedling discrimination were also evaluated. Two different sensors, a still visible camera and a six-band multispectral camera, and three flight altitudes (30, 60 and 100 m) were tested over a naturally infested sunflower field. The main phases of the UAV workflow were the following: 1) mission planning, 2) UAV flight and image acquisition, and 3) image pre-processing. Three different aspects were needed to plan the route: flight area, camera specifications and UAV tasks. The pre-processing phase included the correct alignment of the six bands of the multispectral imagery and the orthorectification and mosaicking of the individual images captured in each flight. The image pixel size, area covered by each image and flight timing were very sensitive to flight altitude. At a lower altitude, the UAV captured images of finer spatial resolution, although the number of images needed to cover the whole field may be a limiting factor due to the energy required for a greater flight length and computational requirements for the further mosaicking process. Spectral differences between weeds, crop and bare soil were significant in the vegetation indices studied (Excess Green Index, Normalised Green-Red Difference Index and Normalised Difference Vegetation Index), mainly at a 30 m altitude. However, greater spectral separability was obtained between vegetation and bare soil with the index NDVI. These results suggest that an agreement among spectral and spatial resolutions is needed to optimise the flight mission according to every agronomical objective as affected by the size of the smaller object to be discriminated (weed plants or weed patches).

  9. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    Science.gov (United States)

    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.

  10. Human in vivo regional intestinal permeability: quantitation using site-specific drug absorption data.

    Science.gov (United States)

    Sjögren, Erik; Dahlgren, David; Roos, Carl; Lennernäs, Hans

    2015-06-01

    Application of information on regional intestinal permeability has been identified as a key aspect of successful pharmaceutical product development. This study presents the results and evaluation of an approach for the indirect estimation of site-specific in vivo intestinal effective permeability (Peff) in humans. Plasma concentration-time profiles from 15 clinical studies that administered drug solutions to specific intestinal regions were collected and analyzed. The intestinal absorption rate for each drug was acquired by deconvolution, using historical intravenous data as reference, and used with the intestinal surface area and the dose remaining in the lumen to estimate the Peff. Forty-three new Peff values were estimated (15 from the proximal small intestine, 11 from the distal small intestine, and 17 from the large intestine) for 14 active pharmaceutical ingredients representing a wide range of biopharmaceutical properties. A good correlation (r(2) = 0.96, slope = 1.24, intercept = 0.030) was established between these indirect jejunal Peff estimates and jejunal Peff measurements determined directly using the single-pass perfusion double balloon technique. On average, Peff estimates from the distal small intestine and large intestine were 90% and 40%, respectively, of those from the proximal small intestine. These results support the use of the evaluated deconvolution method for indirectly estimating regional intestinal Peff in humans. This study presents the first comprehensive data set of estimated human regional intestinal permeability values for a range of drugs. These biopharmaceutical data can be used to improve the accuracy of gastrointestinal absorption predictions used in drug development decision-making.

  11. Evaluating the results of a site-specific PSHA from the perspective of a risk analyst

    Science.gov (United States)

    Klügel, Jens-Uwe

    2016-04-01

    From 1998 till 2015 Swiss Nuclear Power Plants sponsored a set of comprehensive site-specific PSHA-studies (PEGASOS, PEGASOS Refinement Project) to obtain the requested input for their plant specific probabilistic risk assessments following the US SSHAC procedures at their most elaborated level 4. The studies were performed by well-known earth scientists working completely independent from sponsors under participatory review of the Swiss Nuclear Safety Inspectorate. Risk analysts of Swiss Nuclear Power Plants recently have been mandated to implement the final results of the studies in their risk assessment studies. This triggered an in depth assessment of the results focussed on their practical applicability for risk studies. This assessment resulted in some important insights that are of interest for future PSHA studies performed for new nuclear power plants. The assessment included a review of the completeness of results with respect to risk applications as well as plausibility checks of hazard results based on Black Swan Theory and known historical events. The key lessons and recommendations for more detailed project output specifications for future projects are presented in the paper. It was established that future PSHA projects shall provide the joint probability distribution of ground motion hazard and the associated strong motion duration as the output to allow for a technically meaningful risk assessment. The recommendation of WENRA (West European Nuclear Regulators) published in their reference levels to perform natural hazard assessment preferably based on physical grounds (deterministic method) is also rationalized by recommending an holistic approach to hazard analysis comparing PSHA insights with the results of modelling deterministic Seismic Hazard Analysis.

  12. Configuration and specifications of an Unmanned Aerial Vehicle (UAV for early site specific weed management.

    Directory of Open Access Journals (Sweden)

    Jorge Torres-Sánchez

    Full Text Available A new aerial platform has risen recently for image acquisition, the Unmanned Aerial Vehicle (UAV. This article describes the technical specifications and configuration of a UAV used to capture remote images for early season site- specific weed management (ESSWM. Image spatial and spectral properties required for weed seedling discrimination were also evaluated. Two different sensors, a still visible camera and a six-band multispectral camera, and three flight altitudes (30, 60 and 100 m were tested over a naturally infested sunflower field. The main phases of the UAV workflow were the following: 1 mission planning, 2 UAV flight and image acquisition, and 3 image pre-processing. Three different aspects were needed to plan the route: flight area, camera specifications and UAV tasks. The pre-processing phase included the correct alignment of the six bands of the multispectral imagery and the orthorectification and mosaicking of the individual images captured in each flight. The image pixel size, area covered by each image and flight timing were very sensitive to flight altitude. At a lower altitude, the UAV captured images of finer spatial resolution, although the number of images needed to cover the whole field may be a limiting factor due to the energy required for a greater flight length and computational requirements for the further mosaicking process. Spectral differences between weeds, crop and bare soil were significant in the vegetation indices studied (Excess Green Index, Normalised Green-Red Difference Index and Normalised Difference Vegetation Index, mainly at a 30 m altitude. However, greater spectral separability was obtained between vegetation and bare soil with the index NDVI. These results suggest that an agreement among spectral and spatial resolutions is needed to optimise the flight mission according to every agronomical objective as affected by the size of the smaller object to be discriminated (weed plants or weed patches.

  13. Joint analysis of site-specific cancer risks for the atomic bomb survivors

    International Nuclear Information System (INIS)

    Statistical methods are presented for joint analysis of site-specific cancer risks for the atomic-bomb survivors. Previous analyses of these data, aside from those on leukemia, have been made either without regard to cancer type, or separately for types or classes of cancers. Clearly, analyses without regard to cancer type are less than satisfactory. The primary advantages of joint, rather than separate, analyses are that (1) models can be fitted with parameters common to cancer types, which can allow more-precise estimation of effects of interest, (2) significance tests can be used to compare type-specific risks, and (3) a clearer understanding may be obtained of risk-modification factors such as sex, age at exposure, and time since exposure. Joint analysis is straightforward, entailing primarily the incorporation of another factor for cancer type in the usual cross-tabulation of the data for analysis. The use of these methods is illustrated in an analysis of the three categories of cancer studied by the fifth Advisory Committee on the Biological Effects of Ionizing Radiation (BEIR V): digestive, respiratory, and other solid tumors. Based on this analysis, some criticism is made of the BEIR V-preferred models. Since the proposed methods are applicable to models for either relative or absolute risks, some comments on the use of explicit models for the absolute excess risk are also given. Although some of the gains from joint analysis are apparent from the results here, it will be important to use these methods with a more suitable choice of cancer classes and for cancer incidence data in which the diagnoses are more accurate. (author)

  14. A site-specific ecological risk assessment for corn-associated insecticides.

    Science.gov (United States)

    Whiting, Sara A; Lydy, Michael J

    2015-07-01

    A site-specific ecological risk assessment (ERA) was conducted to examine the simultaneous use of genetically modified corn (Bt corn) with a neonicotinoid seed coating, clothianidin, and use of a granular insecticide, tefluthrin, to protect crops from pest damage. A field study was conducted on site, and exposure data from the literature were summarized to determine the matrices and exposure concentrations that nontarget species could typically experience within an agricultural ecosystem. To determine ecological effects on nontarget species, acute toxicity bioassays were conducted on earthworms (Eisenia fetida), amphipods (Hyalella azteca), and Elmid riffle beetle larvae (Ancyronyx spp.) in which the test species were exposed to single insecticides as well as the mixture of the 3 insecticides. In the risk characterization section of the ERA, stressor-response profiles for each species tested were compared with field distributions of the insecticides, and a margin of safety at the 10th percentile (MOS10) was calculated to estimate risk. No acute toxicity was observed in any of the 3 nontarget species after exposure to senescent Bt corn leaf tissue. Large MOS10 values were calculated for clothianidin to the nontarget species. When bioassays were compared with tefluthrin field distributions, very low MOS10 values were calculated for earthworms (0.06) and H. azteca (0.08) because the environmental concentrations often exceeded the stressor-response profile. No increased toxicity was observed when nontarget species were exposed to a mixture of the 3 insecticides. In summary, the genetically modified corn insecticidal proteins and clothianidin were not found at environmental concentrations exceeding benchmark values for ecological effects, but tefluthrin was consistently detected in the environment at levels that could be causing toxicity to nontarget species, especially if this pyrethroid is able to travel off site.

  15. Environmental restoration and waste management site-specific plan for Richland Operations Office

    International Nuclear Information System (INIS)

    This document was prepared to implement and support the US Department of Energy-Headquarters (DOE-HQ) national plan. The national plan, entitled Environmental Restoration and Waste Management Five-Year Plan (DOE 1990b) (hereinafter referred to as the DOE-HQ Five-Year Plan) is the cornerstone of the US Department of Energy's (DOE) long-term strategy in environmental restoration and waste management. The DOE-HQ Five-Year Plan addresses overall philosophy and environmental and waste-related activities under the responsibilities of the DOE Office of Environmental Restoration and Waste Management. The plan also reaffirms DOE-HQ goals to bring its nuclear sites into environmental compliance in cooperation with its regulators and the public, and to clean up and restore the environment by 2019 (the commitment for the Hanford Site is for one year sooner, or 2018). This document is part of the site-specific plan for the US Department of Energy-Richland Operations Office (DOE-RL). It is the first revision of the original plan, which was dated December 1989 (DOE-RL 1989a). This document is a companion document to the Overview of the Hanford Cleanup Five-Year Plan (DOE-RL 1989d) and The Hanford Site Environmental Restoration and Waste Management Five-Year Plan Activity Data Sheets (DOE-RL 1991). Although there are three documents that make up the complete DOE-RL plan, this detailed information volume was prepared so it could be used as a standalone document. 71 refs., 40 figs., 28 tabs

  16. Synthesis and characterization of carboxymethyl chitosan hydrogel: Application as site specific delivery for lercanidipine hydrochloride

    Indian Academy of Sciences (India)

    Subhash S Vaghani; Madhabhai M Patel; C S Satish; Kandarp M Patel; N P Jivani

    2012-12-01

    In the present study, carboxymethylchitosan (CMCS) was prepared from chitosan, crosslinked with glutaraldehyde and evaluated in vitro as a potential carrier for site specific drug delivery of lercanidipine hydrochloride (LERH). LERH was incorporated at the time of crosslinking of CMCS. The chitosan was evaluated for its degree of deacetylation () and average molecular weight, which were found to be 84.6% and 3.5 × 104 Da, respectively. The degree of substitution on prepared CMCS was found to be 0.68. All hydrogel formulations showed more than 86% and 77% yield and drug loading, respectively. The swelling behaviour of prepared hydrogels were checked in different pH values, 1.2, 6.8 and 7.4, indicated pH responsive swelling characteristic with very less swelling at pH 1.2 and quick swelling at pH 6.8 followed by linear swelling at pH 7.4 with slight increase. In vitro release profile was carried out at the same conditions as in swelling and drug release was found to be dependent on swelling of hydrogels and showed biphasic release pattern with non-fickian diffusion kinetics at higher pH. The carboxymethylation of chitosan, entrapment of drug and its interaction in prepared hydrogels were checked by FTIR, 1H-NMR, DSC and -XRD studies, which confirmed formation of CMCS from chitosan and absence of any significant chemical change in LERH after being entrapped in crosslinked hydrogel formulations. The surface morphology of formulation 6 was checked before and after dissolution, revealed open channel like pores formation after dissolution.

  17. Environmental restoration and waste management site-specific plan for Richland Operations Office. [Contains glossary

    Energy Technology Data Exchange (ETDEWEB)

    1991-09-01

    This document was prepared to implement and support the US Department of Energy-Headquarters (DOE-HQ) national plan. The national plan, entitled Environmental Restoration and Waste Management Five-Year Plan (DOE 1990b) (hereinafter referred to as the DOE-HQ Five-Year Plan) is the cornerstone of the US Department of Energy's (DOE) long-term strategy in environmental restoration and waste management. The DOE-HQ Five-Year Plan addresses overall philosophy and environmental and waste-related activities under the responsibilities of the DOE Office of Environmental Restoration and Waste Management. The plan also reaffirms DOE-HQ goals to bring its nuclear sites into environmental compliance in cooperation with its regulators and the public, and to clean up and restore the environment by 2019 (the commitment for the Hanford Site is for one year sooner, or 2018). This document is part of the site-specific plan for the US Department of Energy-Richland Operations Office (DOE-RL). It is the first revision of the original plan, which was dated December 1989 (DOE-RL 1989a). This document is a companion document to the Overview of the Hanford Cleanup Five-Year Plan (DOE-RL 1989d) and The Hanford Site Environmental Restoration and Waste Management Five-Year Plan Activity Data Sheets (DOE-RL 1991). Although there are three documents that make up the complete DOE-RL plan, this detailed information volume was prepared so it could be used as a standalone document. 71 refs., 40 figs., 28 tabs.

  18. Site-specific lead exposure from lead pellet ingestion in sentinel mallards

    Science.gov (United States)

    Rocke, T.E.; Brand, C.J.; Mensik, John G.

    1997-01-01

    We monitored lead poisoning from the ingestion of spent lead pellets in sentinel mallards (Anas platyhrynchos) at the Sacramento National Wildlife Refuge (SNWR), Willows, California for 4 years (1986-89) after the conversion to steel shot for waterfowl hunting on refuges in 1986. Sentinel mallards were held in 1.6-ha enclosures in 1 hunted (P8) and 2 non-hunted (T19 and TF) wetlands. We compared site-specific rates of lead exposure, as determined by periodic measurement of blood lead concentrations, and lead poisoning mortality between wetlands with different lead pellet densities, between seasons, and between male and female sentinels. In 1986, the estimated 2-week rate of lead exposure was significantly higher (P 2,000,000 pellets/ha), than in those with lower densities of lead pellets, T19 (18.1%; 173,200 pellets/ha) and TF (0.9%; 15,750 pellets/ha). The probability of mortality from lead poisoning was also significantly higher (P < 0.01) in sentinel mallards enclosed in P8 (0.25) than T19 (0) and TF (0) in 1986 and remained significantly higher (P < 0.001) during the 4-year study. Both lead exposure and the probability of lead poisoning mortality in P8 were significantly higher (P < 0.001) in the fall of 1986 (43.8%; 0.25), before hunting season, than in the spring of 1987 (21.6%; 0.04), after hunting season. We found no significant differences in the rates of lead exposure or lead poisoning mortality between male and female sentinel mallards. The results of this study demonstrate that in some locations, lead exposure and lead poisoning in waterfowl will continue to occur despite the conversion to steel shot for waterfowl hunting.

  19. Site-specific genomic (SSG and random domain-localized (RDL mutagenesis in yeast

    Directory of Open Access Journals (Sweden)

    Honigberg Saul M

    2004-04-01

    Full Text Available Abstract Background A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. Results We have modified the existing method for introducing deletions in the yeast (S. cerevisiae genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis or is synthesized by error-prone PCR (RDL mutagenesis. In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. Conclusion SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

  20. Site-specific differences of insulin action in adipose tissue derived from normal prepubertal children

    International Nuclear Information System (INIS)

    Body fat distribution determines obesity-related morbidity in adults but little is known of the aetiology or pathophysiology in children. This study investigates differences in insulin-mediated metabolism in primary cell cultures of subcutaneous and visceral preadipocytes derived from prepubertal children. The impact of differentiation and responses to TNFα exposure was also investigated. Proliferation rates were greater in subcutaneous versus visceral preadipocytes (41 h(3) versus 69 h(4); P = 0.008). Insulin caused a dose-dependent increase in GSK-3 phosphorylation and an increase in MAPK phosphorylation over time, with increased sensitivity in subcutaneous preadipocytes. Post-differentiation, dose-dependent increases in GSK-3 phosphorylation were maintained, while MAPK phosphorylation was identical in both subtypes. No changes were observed in insulin receptor abundance pre-/post-differentiation. GLUT4 abundance was significantly increased in visceral versus subcutaneous adipocytes by 76(4)%; P = 0.03), coincidental with increased insulin-stimulated 2-deoxy-glucose transport (+150(26)% versus +79(10)%; P = 0.014) and further elevated by acute exposure to TNFα (+230(52)%; P = 0.019 versus +123(24)%; P = 0.025, respectively). TNFα also significantly increased basal glucose transport rates (+44(14)%; P = 0.006 versus +34(11)%; P = 0.007) and GLUT1 localisation to the plasma membrane. These data establish site-specific differences in subcutaneous and visceral fat cells from children. Responses to insulin varied with differentiation and TNFα exposure in the two depots, consistent with parallel changes in GLUT1/4 abundance and localisation

  1. Site-specific DICER and DROSHA RNA products control the DNA-damage response.

    Science.gov (United States)

    Francia, Sofia; Michelini, Flavia; Saxena, Alka; Tang, Dave; de Hoon, Michiel; Anelli, Viviana; Mione, Marina; Carninci, Piero; d'Adda di Fagagna, Fabrizio

    2012-08-01

    Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

  2. Configuration and specifications of an Unmanned Aerial Vehicle (UAV) for early site specific weed management.

    Science.gov (United States)

    Torres-Sánchez, Jorge; López-Granados, Francisca; De Castro, Ana Isabel; Peña-Barragán, José Manuel

    2013-01-01

    A new aerial platform has risen recently for image acquisition, the Unmanned Aerial Vehicle (UAV). This article describes the technical specifications and configuration of a UAV used to capture remote images for early season site- specific weed management (ESSWM). Image spatial and spectral properties required for weed seedling discrimination were also evaluated. Two different sensors, a still visible camera and a six-band multispectral camera, and three flight altitudes (30, 60 and 100 m) were tested over a naturally infested sunflower field. The main phases of the UAV workflow were the following: 1) mission planning, 2) UAV flight and image acquisition, and 3) image pre-processing. Three different aspects were needed to plan the route: flight area, camera specifications and UAV tasks. The pre-processing phase included the correct alignment of the six bands of the multispectral imagery and the orthorectification and mosaicking of the individual images captured in each flight. The image pixel size, area covered by each image and flight timing were very sensitive to flight altitude. At a lower altitude, the UAV captured images of finer spatial resolution, although the number of images needed to cover the whole field may be a limiting factor due to the energy required for a greater flight length and computational requirements for the further mosaicking process. Spectral differences between weeds, crop and bare soil were significant in the vegetation indices studied (Excess Green Index, Normalised Green-Red Difference Index and Normalised Difference Vegetation Index), mainly at a 30 m altitude. However, greater spectral separability was obtained between vegetation and bare soil with the index NDVI. These results suggest that an agreement among spectral and spatial resolutions is needed to optimise the flight mission according to every agronomical objective as affected by the size of the smaller object to be discriminated (weed plants or weed patches). PMID:23483997

  3. Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway▿

    OpenAIRE

    Daya, Shyam; Cortez, Nenita; Berns, Kenneth I.

    2009-01-01

    Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two prot...

  4. Site-specific rectocele repair with dermal graft augmentation: comparison of porcine dermal xenograft (Pelvicol) and human dermal allograft.

    Science.gov (United States)

    Biehl, Roger C; Moore, Robert D; Miklos, John R; Kohli, Neeraj; Anand, Indu S; Mattox, T Fleming

    2008-01-01

    This study is a retrospective chart review comparing 195 women who underwent rectocele repair with either a porcine dermal xenograft or human allogenic cadaveric dermal graft augmentation over a two year period. A site-specific defect repair was completed prior to augmentation with the graft. Examinations were performed preoperatively and postoperatively using the pelvic organ prolapse quantification system. Questionnaires were used to assess constipation and dyspareunia. De novo dyspareunia and cure rates for constipation and dyspareunia were not statistically different between the two groups. Site-specific fascial rectocele repairs with xenograft or allograft augmentation were found to have similar complication rates as well as objective and subjective cure rates.

  5. Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

    Science.gov (United States)

    Baumann, Fabian; Bauer, Magnus S.; Milles, Lukas F.; Alexandrovich, Alexander; Gaub, Hermann E.; Pippig, Diana A.

    2016-01-01

    Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

  6. Cysteine-containing peptide tag for site-specific conjugation of proteins

    Science.gov (United States)

    Backer, Marina V.; Backer, Joseph M.

    2008-04-08

    The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

  7. Evaluation and interpretation of regional and site-specific hydrochemical data bases for water quality assessment

    Directory of Open Access Journals (Sweden)

    M. G. Hutchins

    1999-01-01

    Full Text Available The spatial distribution of stream water composition, as determined by the Geochemical Baseline Survey of the Environment (G-BASE conducted by the British Geological Survey (BGS can be successfully related under baseflow conditions to bedrock geochemistry. Further consideration of results in conjunction with site-specific monitoring data enables factors controlling both spatial and temporal variability in major element composition to be highlighted and allows the value of the survey to be enhanced. Hence, chemical data (i from streams located on Lower Silurian (Llandovery bedrock at 1 km2 resolution collected as part of the G-BASE survey of Wales and the West Midlands and (ii from catchment monitoring studies located in upland mid-Wales (conducted by Institute of Terrestrial Ecology, have been considered together as an example. Classification of the spatial survey data set in terms of potentially controlling factors was carried out so as to illustrate the level of explanation they could give in terms of observed spatial chemical variability. It was therefore hypothesised that on a geological lithostratigraphic series of limited geochemical contrast, altitude and land-use factors provide better explanation of this variability than others such as lithology at sampling site and stream order. At an individual site, temporal variability was also found to be of considerable significance and, at a monthly time-step, is explicable in terms of factors such as antecedent conditions and seasonality. Data suggest that the degree of this variability may show some relationship with stream order and land-use. Monitoring data from the region also reveal that relationships between stream chemistry and land-use may prove to be strong not only at base flow but also in storm flow conditions. In a wider context, predictions of the sensitivity of stream water to acidification based on classifications of soil and geology are successful on a regional scale. However

  8. Site-specific hyperphosphorylation of pRb in HIV-induced neurotoxicity.

    Science.gov (United States)

    Akay, C; Lindl, K A; Wang, Y; White, M G; Isaacman-Beck, J; Kolson, D L; Jordan-Sciutto, K L

    2011-06-01

    nonphosphorylatable pRb (ppRb S795A) attenuated HIV-MDM-induced neurotoxicity. These findings indicate that HIV-infection in the brain is associated with site-specific hyperphosphorylation of pRb at serine795, which is not induced by other tested stimuli, and that this phosphorylation contributes to neuronal death in this disease, demonstrating that specific pRb sites are differentially targeted and may have diverse impacts on the viability of post-mitotic neurons.

  9. Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

    International Nuclear Information System (INIS)

    Full text: Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125I in a plasmid bound by a 125I-labeled triplex forming oligonucleotide (125 I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125 I target base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the

  10. Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

    International Nuclear Information System (INIS)

    Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125I in a plasmid bound by a 125I-labeled triplex forming oligonucleotide ( 125I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125Itarget base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the absence of

  11. Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

    Directory of Open Access Journals (Sweden)

    Ruley H Earl

    2004-10-01

    Full Text Available Abstract Background Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells. Results In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6. The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes, and did not require localization of the enzyme to the nucleus. Conclusions The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.

  12. Controlled, Site-Specific Functionalization of Carbon Nanotubes with Diazonium Salts

    Science.gov (United States)

    Tour, James M.

    2013-01-01

    capacities and uses. Site-specific functionalization may enable the use of nanotubes in molecular electronic applications because device functionality is critical at the cross points.

  13. Spatial and temporal precipitation variability as a component of site-specific crop yield variability

    Science.gov (United States)

    O'Neal, Monte Ray

    The purpose of this study was to determine spatial and temporal precipitation variability, and the effect of this variability on yield and profitability. On-farm precipitation data is currently being measured by site- specific farmers. One potential use of the data is to provide inputs for corn yield modeling, which has been performed with neural networks and simulation. Profitability of measuring on-farm data depends on spatial precipitation variability and its effect on yield. Precipitation and air temperature from corn silking to dent stages, scale of yield data, and a technology factor were used to model corn yield in east central Indiana at farm (250 ha), county, and state scales, using backpropagation neural networks with five data coding schemes. The best net gave a root-mean-squared error of 11.9% overall (10.9% farm, 10.5% county, 7.9% state yield), with maximum-value interval coding. Four rain gauges on the same farm, spaced apart 1.02 to 3.04 km, gave a median absolute deviation of precipitation among gauges, by corn and soybean phenological phase, of 0.25 to 1.73 mm.day-1 (spatial variability). Median absolute deviation from a reference year was 0.17 to 3.40 mm.day-1 (temporal/year-to- year variability). Spatial variability was less than temporal variability, and frequently less than 1 mm. Three precipitation data sources-a National Weather Service (NWS) station on the same farm, the nearest non- urban NWS station, and a weighted mean of three nearest non-urban NWS stations (27-35 km distance)-were used to simulate corn yield by 1-ha grid cells with CERES-Maize. The percent absolute difference of simulated yield among the three sources (effect of spatial precipitation variability) was 15.8%. The percent absolute difference from long-term mean (temporal variability) was 21.5%, of the same order as spatial variability. A choice among nitrogen application strategies-variable-rate versus whole-field application, starter versus no starter-was made for the

  14. Site-Specific Nutrient Management for Rainfed Maize in Western Mid-Hills of Nepal

    Directory of Open Access Journals (Sweden)

    Prakash Ghimire

    2015-06-01

    Full Text Available On-farm experiment was conducted in eight farmers’ field, of Khasyoli village development committee (952 to 1415 masl, Nepal, from April to September, 2011 to address the major constraint (nutrient management to maize production through site-specific nutrient management (SSNM as this approach is popular among scientists. The experiment comprised three nutrient omission plots (0N, 0P, and 0K, an ample NPK plot, and a farmers’ fertilization practice (FFP plot, arranged in randomized complete block design. Farmers planted open pollinated variety (Manakamana-3 and managed in their way. Field-specific NPK application rates were calculated by considering nutrient demand, indigenous NPK supply and recovery efficiency of fertilizers. Grain yield in FFP (2.32 Mg/ha and 0N (1.79 Mg/ha plots differed significantly from each other and rest of the treatments, but was statistically similar among 0P (3.18 Mg/ha, 0K (3.40 Mg/ha and ample NPK (3.38 Mg/ha plots. Post-harvest grain and stover analysis revealed that indigenous NPK supply (20-71 kg N, 19-68 kg P2O5 and 51-164 kg K2O/ha of soil vary among the farmers’ field. Moreover, soil was poor in indigenous N supply (42 kg/ha, but rich in indigenous P2O5 (35 kg/ha and K2O (90 kg/ha supply, on an average. As per the principles of SSNM, the initial fertilizer recommendation made can vary from 40-222 kg N, 0- 93 kg P2O5, and 0-50 kg K2O/ha. On an average, farmers may apply no or lower dose of P2O5 (18 kg/ha and K2O (3 kg/ha but need to significantly increase dose of N (143 kg/ha fertilizer for enhancing soil and maize productivity.

  15. Site-specific tolerance tables and indexing device to improve patient setup reproducibility.

    Science.gov (United States)

    McCullough, Kiernan T; James, Joshua A; Cetnar, Ashley J; McCullough, Mark A; Wang, Brian

    2015-01-01

    While the implementation of tools such as image-guidance and immobilization devices have helped to prevent geometric misses in radiation therapy, many treatments remain prone to error if these items are not available, not utilized for every fraction, or are misused. The purpose of this project is to design a set of site-specific treatment tolerance tables to be applied to the treatment couch for use in a record and verify (R&V) system that will insure accurate patient setup with minimal workflow interruption. This project also called for the construction of a simple indexing device to help insure reproducible patient setup for patients that could not be indexed with existing equipment. The tolerance tables were created by retrospective analysis on a total of 66 patients and 1,308 treatments, separating them into five categories based on disease site: lung, head and neck (H&N), breast, pelvis, and abdomen. Couch parameter tolerance tables were designed to encompass 95% of treatments, and were generated by calculating the standard deviation of couch vertical, longitudinal, and lateral values using the first day of treatment as a baseline. We also investigated an alternative method for generating the couch tolerances by updating the baseline values when patient position was verified with image guidance. This was done in order to adapt the tolerances to any gradual changes in patient setup that would not correspond with a mistreatment. The tolerance tables and customizable indexing device were then implemented for a trial period in order to determine the feasibility of the system. During this trial period we collected data from 1,054 fractions from 65 patients. We then analyzed the number of treatments that would have been out of tolerance, as well as whether or not the tolerances or setup techniques should be adjusted. When the couch baseline values were updated with every imaging fraction, the average rate of tolerance violations was 10% for the lung, H&N, abdomen

  16. Textpresso Site-Specific Recombinases: a text-mining server for the recombinase literature including Cre mice and conditional alleles

    Science.gov (United States)

    Urbanski, William M.; Condie, Brian G.

    2016-01-01

    Textpresso Site Specific Recombinases (http://ssrc.genetics.uga.edu/) is a text-mining web server for searching a database of over 9000 full-text publications. The papers and abstracts in this database represent a wide range of topics related to site-specific recombinase (SSR) research tools. Included in the database are most of the papers that report the characterization or use of mouse strains that express Cre recombinase as well as papers that describe or analyze mouse lines that carry conditional (floxed) alleles or SSR-activated transgenes/knockins. The database also includes reports describing SSR-based cloning methods such as the Gateway or the Creator systems, papers reporting the development or use of SSR-based tools in systems such as Drosophila, bacteria, parasites, stem cells, yeast, plants, zebrafish and Xenopus as well as publications that describe the biochemistry, genetics or molecular structure of the SSRs themselves. Textpresso Site Specific Recombinases is the only comprehensive text-mining resource available for the literature describing the biology and technical applications of site-specific recombinases. PMID:19882667

  17. Chironomid-based water depth reconstructions: an independent evaluation of site-specific and local inference models

    NARCIS (Netherlands)

    S. Engels; L.C. Cwynar; A.B.H. Rees; B.N. Shuman

    2012-01-01

    Water depth is an important environmental variable that explains a significant portion of the variation in the chironomid fauna of shallow lakes. We developed site-specific and local chironomid water-depth inference models using 26 and 104 surface-sediment samples, respectively, from seven kettlehol

  18. A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing.

    Science.gov (United States)

    Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B; Goth, Christoffer K; Petersen, Thomas Nordahl; Brunak, Søren; Bennett, Eric P; Levery, Steven B; Clausen, Henrik

    2011-11-18

    Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.

  19. Sensing Site-Specific Variability in Soil and Plant Phosphorus and Other Mineral Nutrients by X-Ray Fluorescence Spectrometry

    Science.gov (United States)

    Detection and rapid response to in-season changes of soil nutrient availability and plant needs with weather conditions and site-specific characteristics are essential to the optimal performance of an agronomic crop production system. With recent advances in material science, detector design and se...

  20. A novel bench-scale column assay to investigate site-specific nitrification biokinetics in biological rapid sand filters

    DEFF Research Database (Denmark)

    Tatari, Karolina; Smets, Barth F.; Albrechtsen, Hans-Jørgen

    2013-01-01

    A bench-scale assay was developed to obtain site-specific nitrification biokinetic information from biological rapid sand filters employed in groundwater treatment. The experimental set-up uses granular material subsampled from a full-scale filter, packed in a column, and operated with controlled...

  1. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup;

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  2. Local nutrient regimes determine site-specific environmental triggers of cyanobacterial and microcystin variability in urban lakes

    Science.gov (United States)

    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.

  3. 44 CFR 354.5 - Description of site-specific, plume pathway EPZ biennial exercise-related component services and...

    Science.gov (United States)

    2010-10-01

    ... 44 Emergency Management and Assistance 1 2010-10-01 2010-10-01 false Description of site-specific... Emergency Management and Assistance FEDERAL EMERGENCY MANAGEMENT AGENCY, DEPARTMENT OF HOMELAND SECURITY... exercise logistics. (4) Conduct plume pathway EPZ biennial exercises, evaluations, and post...

  4. A Free-Radical-Promoted Site-Specific Cross-Dehydrogenative-Coupling of N-Heterocycles with Fluorinated Alcohols.

    Science.gov (United States)

    Xu, Zhengbao; Hang, Zhaojia; Chai, Li; Liu, Zhong-Quan

    2016-09-16

    A C-C formation of an electron-rich N-heterocycle with fluorinated alcohol is developed. Through this radical-triggered cross-dehydrogenative coupling strategy, a wide range of useful building blocks such as C3 hydroxyfluoroalkylated indoles and pyrroles can be site-specifically synthesized. Mechanistic studies indicate a single-electron-transfer initiated radical cycle would be involved.

  5. Local nutrient regimes determine site-specific environmental triggers of cyanobacterial and microcystin variability in urban lakes

    Directory of Open Access Journals (Sweden)

    S. C. Sinang

    2014-10-01

    Full Text Available 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.

  6. Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria.

    Science.gov (United States)

    Cui, Lun; Shearwin, Keith E

    2017-01-01

    Clonetegration is a method for site-specific insertion of DNA into prokaryotic chromosomes, based on bacteriophage integrases. The method combines DNA cloning/assembly and chromosomal integration into a single step, providing a simple and rapid strategy for inserting DNA sequences into bacterial chromosomes. PMID:27671938

  7. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    Science.gov (United States)

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  8. Comparing measured and modelled soil carbon: which site-specific variables are linked to high stability?

    Science.gov (United States)

    Robertson, Andy; Schipanski, Meagan; Ma, Liwang; Ahuja, Lajpat; McNamara, Niall; Smith, Pete; Davies, Christian

    2016-04-01

    Changes in soil carbon (C) stocks have been studied in depth over the last two decades, as net greenhouse gas (GHG) sinks are highlighted to be a partial solution to the causes of climate change. However, the stability of this soil C is often overlooked when measuring these changes. Ultimately a net sequestration in soils is far less beneficial if labile C is replacing more stable forms. To date there is no accepted framework for measuring soil C stability, and as a result there is considerable uncertainty associated with the simulated impacts of land management and land use change when using process-based systems models. However, a recent effort to equate measurable soil C fractions to model pools has generated data that help to assess the impacts of land management, and can ultimately help to reduce the uncertainty of model predictions. Our research compiles this existing fractionation data along with site metadata to create a simplistic statistical model able to quantify the relative importance of different site-specific conditions. Data was mined from 23 published studies and combined with original data to generate a dataset of 100+ land use change sites across Europe. For sites to be included they required soil C fractions isolated using the Zimmermann et al. (2007) method and specific site metadata (mean annual precipitation, MAP; mean annual temperature, MAT; soil pH; land use; altitude). Of the sites, 75% were used to develop a generalized linear mixed model (GLMM) to create coefficients where site parameters can be used to predict influence on the measured soil fraction C stocks. The remaining 25% of sites were used to evaluate uncertainty and validate this empirical model. Further, four of the aforementioned sites were used to simulate soil C dynamics using the RothC, DayCent and RZWQM2 models. A sensitivity analysis (4096 model runs for each variable applying Latin hypercube random sampling techniques) was then used to observe whether these models place

  9. Theory of site-specific interactions of the combinatorial transcription factors with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Murugan, R, E-mail: rmurugan@gmail.co [Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamil Nadu 600036 (India)

    2010-05-14

    We derive a functional relationship between the mean first passage time associated with the concurrent binding of multiple transcription factors (TFs) at their respective combinatorial cis-regulatory module sites (CRMs) and the number n of TFs involved in the regulation of the initiation of transcription of a gene of interest. Our results suggest that the overall search time {tau}{sub s} that is required by the n TFs to locate their CRMs which are all located on the same DNA chain scales with n as {tau}{sub s{approx}}n{sup {alpha}}where {alpha} {approx} (2/5). When the jump size k that is associated with the dynamics of all the n TFs along DNA is higher than that of the critical jump size k{sub c} that scales with the size of DNA N as k{sub c} {approx} N{sup 2/3}, we observe a similar power law scaling relationship and also the exponent {alpha}. When k < k{sub c}, {alpha} shows a strong dependence on both n and k. Apparently there is a critical number of combinatorial TFs n{sub c} {approx} 20 that is required to efficiently regulate the initiation of transcription of a given gene below which (2/5) < {alpha} < 1 and beyond which {alpha} > 1. These results seem to be independent of the initial distances between the TFs and their corresponding CRMs and also suggest that the maximum number of TFs involved in a given combinatorial regulation of the initiation of transcription of a gene of interest seems to be restricted by the degree of condensation of the genomic DNA. The optimum number m{sub opt} of roadblock protein molecules per genome at which the search time associated with these n TFs to locate their binding sites is a minimum seems to scale as m{sub opt{approx}}Ln{sup {alpha}/2} where L is the sliding length of TFs whose maximum value seems to be such that L {<=} 10{sup 4} bps for the E. coli bacterial genome.

  10. Vascular nanomedicine: Site specific delivery of elastin stabilizing therapeutics to damaged arteries

    Science.gov (United States)

    Sinha, Aditi

    Elastin, a structural protein in the extra-cellular matrix, plays a critical role in the normal functioning of blood vessels. Apart from performing its primary function of providing resilience to arteries, it also plays major role in regulating cell-cell and cell-matrix interactions, response to injury, and morphogenesis. Medial arterial calcification (MAC) and abdominal aortic aneurysm (AAA) are two diseases where the structural and functional integrity of elastin is severely compromised. Although the clinical presentation of MAC and AAA differ, they have one common underlying causative mechanism---pathological degradation of elastin. Hence prevention of elastin degradation in the early stages of MAC and AAA can mitigate, partially if not wholly, the fatal consequences of both the diseases. The work presented here is motivated by the overwhelming statistics of people afflicted by elastin associated cardiovascular diseases and the unavailability of cure for the same. Overall goal of our research is to understand role of elastin degradation in cardiovascular diseases and to develop a targeted vascular drug delivery system that is minimally invasive, biodegradable, and non-toxic, that prevents elastin from degradation. Our hope is that such treatment will also help regenerate elastin, thereby providing a multi-fold treatment option for elasto-degenerative vascular diseases. For this purpose, we have first confirmed the combined role of degraded elastin and hyperglycemia in the pathogenesis of MAC. We have shown that in the absence of degraded elastin and TGF-beta1 (abundantly present in diabetic arteries) vascular smooth muscle cells maintain their homeostatic state, regardless of environmental glucose concentrations. However simultaneous exposure to glucose, elastin peptides and TGF-beta1 causes the pathological transgenesis of vascular cells to osteoblast-like cells. We show that plant derived polyphenols bind to vascular elastin with great affinity resulting in

  11. A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B.; Goth, Christoffer K.;

    2011-01-01

    -type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model......Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition...... and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc...

  12. Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling

    Science.gov (United States)

    Motorin, Yuri; Burhenne, Jürgen; Teimer, Roman; Koynov, Kaloian; Willnow, Sophie; Weinhold, Elmar; Helm, Mark

    2011-01-01

    This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNAPhe. LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments. PMID:21037259

  13. Kinetic Studies on the Irreversible Inhibition of Restriction Endonuclease Pst I by Site-Specific Inhibitors

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The irreversible modifying effects on Pst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou,C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB),diisopropyl fluorophosphate (DFP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3'-sulfonate (woodward's reagent K, WRK ) modify the lysine, cysine,serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity of Pst 1. Used with the irreversible inhibition theory,the apparent inhibition rate constant, A and the microcosmic inhibition rate constants, k+0 and k′ +o of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding.Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration of Pst I conformation and then influence the ability of Pst I recognizing and incising DNA specifically.

  14. Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy.

    Science.gov (United States)

    Basom, Edward J; Maj, Michał; Cho, Minhaeng; Thielges, Megan C

    2016-06-21

    Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function.

  15. High performance electrophoresis system for site-specific entrapment of nanoparticles in a nanoarray

    Science.gov (United States)

    Han, Jin-Hee; Lakshmana, Sudheendra; Kim, Hee-Joo; Hass, Elizabeth A.; Gee, Shirley; Hammock, Bruce D.; Kennedy, Ian

    2010-02-01

    A nanoarray, integrated with an electrophoretic system, was developed to trap nanoparticles into their corresponding nanowells. This nanoarray overcomes the complications of losing the function and activity of the protein binding to the surface in conventional microarrays by using minimum amounts of sample. The nanoarray is also superior to other biosensors that use immunoassays in terms of lowering the limit of detection to the femto- or atto-molar level. In addition, our electrophoretic particle entrapment system (EPES) is able to effectively trap the nanoparticles using a low trapping force for a short duration. Therefore, good conditions for biological samples conjugated with particles can be maintained. The channels were patterned onto a bi-layer consisting of a PMMA and LOL coating on conductive indium tin oxide (ITO)-coated glass slide by using e-beam lithography. The suspensions of 170 nm-nanoparticles then were added to the chip that was connected to a positive voltage. On top of the droplet, another ITO-coated-glass slide was covered and connected to a ground terminal. Negatively charged fluorescent nanoparticles (blue emission) were selectively trapped onto the ITO surface at the bottom of the wells by following electric field lines. Numerical modeling was performed by using commercially available software, COMSOL Multiphysics to provide better understanding about the phenomenon of electrophoresis in a nanoarray. Simulation results are also useful for optimally designing a nanoarray for practical applications.

  16. SEQUENTIAL INVESTMENT IN SITE-SPECIFIC CROP MANAGEMENT UNDER OUTPUT PRICE UNCERTAINTY: IMPLICATIONS FOR NITROGEN POLLUTION CONTROL

    OpenAIRE

    Isik, Murat; Khanna, Madhu; Winter-Nelson, Alex

    2000-01-01

    This paper develops an option value model to examine the extent to which output price uncertainty creates incentives to adopt two interrelated components of site-specific technologies sequentially. It analyzes how the impact of uncertainty on the sequential adoption decision differs across heterogeneous soil conditions, and examines the implications of adoption for nitrogen pollution generation and for the design of a cost-share subsidy policy.

  17. Site-Specific Prevalence and Cell Densities of Selected Microbes in the Lower Reproductive Tract of Menstruating Tampon Users

    OpenAIRE

    Rachelle Eusebio; Meyer, Sandy J.; Berg, Ronald W; Hochwalt, Anne E

    2002-01-01

    OBJECTIVE: To assess differences in prevalence and cell densities of enterococci, Gram negative enterics (GNEs), yeast and Staphylococcus aureus among four genital sites and to examine whether the presence of organisms at one site affected the presence of organisms at other sites. METHODS: Swab samples from the perineum, below and above the hymen, and the posterior fornix obtained from 52 tampon users on menstrual cycle day 3 were analyzed for site-specific prevalence and cell densities of mi...

  18. Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system

    OpenAIRE

    Karimova, M.; Abi-Ghanem, J.; N. Berger; Surendranath, V.; Pisabarro, M. T.; Buchholz, F

    2013-01-01

    Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo. However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is di...

  19. Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification

    OpenAIRE

    Jager, M; Nir, E; Weiss, S

    2006-01-01

    An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence r...

  20. Site-Specific Management of Meloidogyne chitwoodi in Idaho Potatoes Using 1,3-Dichloropropene; Approach, Experiences, and Economics.

    Science.gov (United States)

    King, Bradley A; Taberna, John P

    2013-09-01

    Fumigation for nematode management in irrigated potato production systems of Idaho is widely practiced. Soil injection is the only labeled application method for 1,3-dichloropropene that is conventionally applied on a whole-field basis. Plant-parasitic nematode species exhibit spatially variable population densities that provide an opportunity to practice site-specific fumigation to reduce chemical usage and production costs. During 2002 to 2008, 62 fields intended for commercial potato production in eastern Idaho were sampled using a geo-referenced grid sampling system for plant-parasitic nematode population densities. In total, 4,030 grid samples were collected representing nearly 3,200 ha of commercial potato production. Collectively, 73% of the grid samples had Columbia root knot (CRN) (Meloidogyne chitwoodi) population densities below detectable levels. Site-specific fumigation is the practice of varying application rate of a fumigant based on nematode population density. In 2007, 640 ha of potato production were site-specific fumigated for CRN nematode control in eastern Idaho. On average, this practice resulted in a 30% reduction in chemical usage and production cost savings of $209/ha when 1,3-dichloropropene was used as the sole source of nematode suppression. Reductions in usage of 1,3-dichloropropene can exceed 50% if used in combination with a nonfumigant nematicide such as oxamyl. This combination approach can have production cost savings exceeding $200/ha. Based on farm-gate receipts and USDA inspections provided by potato producers from 2001 to 2011, potato tuber yield and quality have not been adversely affected using site-specific fumigation.

  1. A new usage of functionalized oligodeoxynucleotide probe for site-specific modification of a guanine base within RNA

    OpenAIRE

    Onizuka, Kazumitsu; Taniguchi, Yosuke; Sasaki, Shigeki

    2010-01-01

    Site-specific modification of RNA is of great significance to investigate RNA structure, function and dynamics. Recently, we reported a new method for sequence- and cytosine-selective chemical modification of RNA based on the functional group transfer reaction of the 1-phenyl-2-methylydene-1,3-diketone unit of the 6-thioguanosine base incorporated in the oligodeoxynucleotide probe. In this study, we describe that the functionality transfer rate is greatly enhanced and the selectivity is shift...

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

    Science.gov (United States)

    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.

  3. Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections.

    Directory of Open Access Journals (Sweden)

    Janet M Meredith

    Full Text Available Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the Streptomyces phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against Plasmodium yoelii nigeriensis, reducing average parasite intensity by 85%. Similar protection was observed against Plasmodium falciparum in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade.

  4. Site-specific probabilistic seismic hazard analyses for the Idaho National Engineering Laboratory. Volume 1: Final report

    International Nuclear Information System (INIS)

    This report describes and summarizes a probabilistic evaluation of ground motions for the Idaho National Engineering Laboratory (INEL). The purpose of this evaluation is to provide a basis for updating the seismic design criteria for the INEL. In this study, site-specific seismic hazard curves were developed for seven facility sites as prescribed by DOE Standards 1022-93 and 1023-96. These sites include the: Advanced Test Reactor (ATR); Argonne National Laboratory West (ANL); Idaho Chemical Processing Plant (ICPP or CPP); Power Burst Facility (PBF); Radioactive Waste Management Complex (RWMC); Naval Reactor Facility (NRF); and Test Area North (TAN). The results, probabilistic peak ground accelerations and uniform hazard spectra, contained in this report are not to be used for purposes of seismic design at INEL. A subsequent study will be performed to translate the results of this probabilistic seismic hazard analysis to site-specific seismic design values for the INEL as per the requirements of DOE Standard 1020-94. These site-specific seismic design values will be incorporated into the INEL Architectural and Engineering Standards

  5. Symptomatic and quality of life outcomes after site-specific fascial reattachment for pelvic organ prolapse repair.

    Science.gov (United States)

    Fayyad, Abdalla M; Redhead, Emma; Awan, Noveen; Kyrgiou, Maria; Prashar, Sanjeev; Hill, Simon R

    2008-02-01

    The aim of this study was to assess symptomatic and quality of life outcome scores following site specific fascial reattachment surgery for pelvic organ prolapse using the validated Prolapse Quality of Life (P-QOL) questionnaires. One hundred and ninety two women underwent surgery for pelvic organ prolapse; ninety four underwent anterior repair (thirty four of them had vaginal hysterectomy), and ninety eight had posterior repair. Patients filled P-QOL questionnaires 24 hours prior to surgery and a postal P-QOL questionnaire six months post operatively. Pre and post operative questionnaires were paired. Quality of life and symptoms scores were calculated using Wilcoxon signed rank test. One hundred and one women returned their questionnaires and were suitable to include in the study. Forty nine underwent anterior repair (fifteen had vaginal hysterectomy) and 52 underwent posterior repair. Quality of life scores showed significant improvement in the anterior and posterior repair groups with the exception of general health in the anterior repair group and general health and prolapse impact in the posterior repair group. Anterior repair significantly improved urinary voiding and storage symptoms. Posterior repair group showed significant improvement in defecatory symptoms. Both groups showed improvement in sexual function and general prolapse symptoms. Prolapse repair with site specific fascial reattachment results in significant improvement in quality of life scores six months after surgery. Anterior repair improves urinary voiding and storage symptoms and posterior repair improves defecatory dysfunction and urinary voiding. Sexual function improves following prolapse repair with site specific fascial reattachment.

  6. Site-specific probabilistic seismic hazard analyses for the Idaho National Engineering Laboratory. Volume 1: Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-05-01

    This report describes and summarizes a probabilistic evaluation of ground motions for the Idaho National Engineering Laboratory (INEL). The purpose of this evaluation is to provide a basis for updating the seismic design criteria for the INEL. In this study, site-specific seismic hazard curves were developed for seven facility sites as prescribed by DOE Standards 1022-93 and 1023-96. These sites include the: Advanced Test Reactor (ATR); Argonne National Laboratory West (ANL); Idaho Chemical Processing Plant (ICPP or CPP); Power Burst Facility (PBF); Radioactive Waste Management Complex (RWMC); Naval Reactor Facility (NRF); and Test Area North (TAN). The results, probabilistic peak ground accelerations and uniform hazard spectra, contained in this report are not to be used for purposes of seismic design at INEL. A subsequent study will be performed to translate the results of this probabilistic seismic hazard analysis to site-specific seismic design values for the INEL as per the requirements of DOE Standard 1020-94. These site-specific seismic design values will be incorporated into the INEL Architectural and Engineering Standards.

  7. Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry.

    Science.gov (United States)

    Someya, Tatsuhiko; Ando, Ami; Kimoto, Michiko; Hirao, Ichiro

    2015-08-18

    Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.

  8. Virus Capsids as Targeted Nanoscale Delivery Vessels of Photoactive Compounds for Site-Specific Photodynamic Therapy

    Science.gov (United States)

    Cohen, Brian A.

    The research presented in this work details the use of a viral capsid as an addressable delivery vessel of photoactive compounds for use in photodynamic therapy. Photodynamic therapy is a treatment that involves the interaction of light with a photosensitizing molecule to create singlet oxygen, a reactive oxygen species. Overproduction of singlet oxygen in cells can cause oxidative damage leading to cytotoxicity and eventually cell death. Challenges with the current generation of FDA-approved photosensitizers for photodynamic therapy primarily stem from their lack of tissue specificity. This work describes the packaging of photoactive cationic porphyrins inside the MS2 bacteriophage capsid, followed by external modification of the capsid with cancer cell-targeting G-quadruplex DNA aptamers to generate a tumor-specific photosensitizing agent. First, a cationic porphyrin is loaded into the capsids via nucleotide-driven packaging, a process that involves charge interaction between the porphyrin and the RNA inside the capsid. Results show that over 250 porphyrin molecules associate with the RNA within each MS2 capsid. Removal of RNA from the capsid severely inhibits the packaging of the cationic porphyrins. Porphyrin-virus constructs were then shown to photogenerate singlet oxygen, and cytotoxicity in non-targeted photodynamic treatment experiments. Next, each porphyrin-loaded capsid is externally modified with approximately 60 targeting DNA aptamers by employing a heterobifunctional crosslinking agent. The targeting aptamer is known to bind the protein nucleolin, a ubiquitous protein that is overexpressed on the cell surface by many cancer cell types. MCF-7 human breast carcinoma cells and MCF-10A human mammary epithelial cells were selected as an in vitro model for breast cancer and normal tissue, respectively. Fluorescently tagged virus-aptamer constructs are shown to selectively target MCF-7 cells versus MCF-10A cells. Finally, results are shown in which porphyrin

  9. Relationship between site-specific loss of thigh muscle and gait performance in women: the HIREGASAKI study.

    Science.gov (United States)

    Abe, Takashi; Ogawa, Madoka; Loenneke, Jeremy P; Thiebaud, Robert S; Loftin, Mark; Mitsukawa, Naotoshi

    2012-01-01

    Sarcopenia is observed as a site-specific loss of skeletal muscle mass, however, it is unknown whether the site-specific sarcopenia is associated with development of physical disability. The purpose of this study was to examine the relationship between age-related thigh muscle loss and gait performance. Fifty-three women aged 52-83 years had their thigh muscle thickness (MTH) measured by ultrasound at five sites on the anterior (30%, 50%, and 70% of thigh length) and posterior (50% and 70% of thigh length) aspects of their thigh. Maximum and normal walking speeds, zig-zag walking time, and maximal voluntary isometric knee extension and flexion strength were measured. Age was inversely correlated to the anterior and posterior MTH ratio (e.g., anterior 50%:posterior 70% MTH ratio [r=-0.426, p=0.002]), thus the site-specific muscle loss of the thigh was observed in the present sample. There were no significant correlations between the anterior/posterior MTH ratio and maximum and normal walking speeds. However, the ratios of anterior 50%:posterior 70% MTH (r=-0.430) and anterior 30%:posterior 70% MTH (r=-0.444) were correlated (p=0.001) to zig-zag walking test. After adjusting for age, height and weight, the anterior 30%:posterior 70% MTH (r=-0.292, p=0.040) was inversely correlated to zig-zag walking performance. Isometric knee extension strength was also inversely correlated to zig-zag walking. Our results suggest that an age-related loss of adductor/quadriceps muscles may be associated with a decrease in a relatively difficult task performance such as zig-zag walking. PMID:22795673

  10. Light-stimulated cargo release from a core–shell structured nanocomposite for site-specific delivery

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Yun; Ling, Li; Li, Xiaofang [Department of Neurology, Affiliated Hospital of Hebei University, Baoding 071000 (China); Chen, Meng [Department of Rheumatology, Affiliated Hospital of Hebei University, Baoding 071000 (China); Su, Likai, E-mail: zhangdong19992003@163.com [Department of Neurology, Affiliated Hospital of Hebei University, Baoding 071000 (China)

    2015-03-15

    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 emission 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.

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

    Science.gov (United States)

    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

  12. Determination of human health risk incorporating experimentally derived site-specific bioaccessibility of arsenic at an old abandoned smelter site.

    Science.gov (United States)

    Yang, Kyung; Im, Jinwoo; Jeong, Seulki; Nam, Kyoungphile

    2015-02-01

    This study was conducted to investigate the contribution of a site-specific bioavailability of arsenic (As) to human health risk at an old abandoned smelter site in Korea. The site was contaminated with As for over 60 years with the same source (As2O3 in flue gas), but concentration and in vitro bioaccessibility (IVBA) of As differed by operable units (OU), which consequently resulted in difference in estimated risk. Soil samples collected from six OUs showed that aqua regia-extractable As concentrations ranged from 9.8 to 52.8mg/kg (average 34.1mg/kg) at OUs 1-5, which had been used as rice paddy field and farmland, and a forest region OU 6 showed much higher As concentrations (14.4-169.8mg/kg, average 85.9mg/kg). IVBA of As, determined from the ratio of Solubility/Bioavailability Research Consortium (SBRC)-extractable As to aqua regia-extractable As had a wide range of values (90th percentile values of 28.2-65.8%). Carcinogenic risk calculated with total soil As concentration was the highest (1.4×10(-4)) at OU 6 and the risk at the other OUs ranged from 3.8×10(-5) to 5.7×10(-5). In contrast, when site-specific relative bioavailability (i.e., IVBA values) was incorporated, the estimated risk was reduced by 29.5-62.0% and the decrease was the highest at OUs 1 and 5 with the lowest IVBA of 28.2%. The results demonstrate that the chemical forms of As may be different although the source of contamination is similar, and site-specific bioavailability affected by the chemical forms is an important factor in determining human health risk. PMID:25490246

  13. Site-specific water quality criteria for lethal/sublethal protection of freshwater fish exposed to zinc in southern Taiwan.

    Science.gov (United States)

    Chen, Wei-Yu; Chen, Tzu-Yin; Hsieh, Nan-Hung; Ju, Yi-Ting

    2016-09-01

    There were considerable concerns about the zinc (Zn) pollution caused by electroplating, chemical, and computer-related high-tech industrial discharges in Kaohsiung Rivers situated at south Taiwan. There is, however, a lack of site-specific water chemistry based toxicity assessment and little is known about the sublethal toxicity on freshwater fish. This study proposes an integrated framework to link experimental and mechanistic model-based data analysis of lethal and sublethal Zn toxicities for grass carp (Ctenopharyn odon idellus) populations for providing the site-specific Zn water quality threshold in Kaohsiung Rivers. A biotic ligand model (BLM) that relates toxicity impairment of physiological active sites impacted by Zn species was used to elucidate the site-specific water chemistry affecting the bioavailability and biological response of grass carp exposed to Zn. Results indicated that 96-h LC50 for mortality and 28-d EC50 for growth inhibition were 474.7 ± 1.3 (mean ± SE) and 149 ± 23.5 μg L(-1), respectively. Here the BLM-based predicted steady-state LC50s for mortality were 2110.7, 818.2, 1303.6, 563.3, and 497.1 μg L(-1), whereas measured steady-state EC50s for growth inhibition were 726.8, 326.2, 373.4, 193.9, and 170.5 μg L(-1) for the Agongdian, Houling, Love, Fengshan, and Gaoping Rivers, respectively. A positive correlation between Mg(2+) and EC50 values were found in both acute (r = 0.94, p water chemistry data and ecophysiological traits would enhance the predictive capacities to assess the potential effect of metal toxicity posed to aquatic organisms. PMID:27337432

  14. Derivation of a chronic site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA.

    Science.gov (United States)

    Brix, Kevin V; DeForest, David K; Cardwell, Rick D; Adams, William J

    2004-03-01

    The purpose of this study was to develop a site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA. The study examined the bioavailability and toxicity of selenium, as selenate, to biota resident to the Great Salt Lake and the potential for dietary selenium exposure to aquatic dependent birds that might consume resident biota. Because of its high salinity, the lake has limited biological diversity with bacteria, algae, diatoms, brine shrimp, and brine flies being the only organisms present in the main (hypersaline) portions of the lake. To evaluate their sensitivity to selenium, a series of acute and chronic toxicity studies were conducted on brine shrimp (Artemia franiciscana), brine fly (Ephydra cinerea), and a hypersaline alga (Dunaliella viridis). The resulting acute and chronic toxicity data indicated that resident species are more selenium tolerant than many freshwater species. Because sulfate is known to reduce selenate bioavailability, this selenium tolerance is thought to result in part from the lake's high ambient sulfate concentrations (>5,800 mg/L). The acute and chronic test results were compared to selenium concentrations expected to occur in a mining effluent discharge located at the south end of the lake. Based on these comparisons, no appreciable risks to resident aquatic biota were projected. Field and laboratory data collected on selenium bioaccumulation in brine shrimp demonstrated a linear relationship between water and tissue selenium concentrations. Applying a dietary selenium threshold of 5 mg/kg dry weight for aquatic birds to this relationship resulted in an estimate of 27 microg/L Se in water as a safe concentration for this exposure pathway and an appropriate chronic site-specific water quality standard. Consequently, protection of aquatic birds represents the driving factor in determining a site-specific water quality standard for selenium. PMID:15285352

  15. An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

    Science.gov (United States)

    Ta, Duy Tien; Redeker, Erik Steen; Billen, Brecht; Reekmans, Gunter; Sikulu, Josephine; Noben, Jean-Paul; Guedens, Wanda; Adriaensens, Peter

    2015-10-01

    In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications. PMID:26243885

  16. A procedure for estimating site specific derived limits for the discharge of radioactive material to the atmosphere

    CERN Document Server

    Hallam, J; Jones, J A

    1983-01-01

    Generalised Derived Limits (GDLs) for the discharge of radioactive material to the atmosphere are evaluated using parameter values to ensure that the exposure of the critical group is unlikely to be underestimated significantly. Where the discharge is greater than about 5% of the GDL, a more rigorous estimate of the derived limit may be warranted. This report describes a procedure for estimating site specific derived limits for discharges of radioactivity to the atmosphere taking into account the conditions of the release and the location and habits of the exposed population. A worksheet is provided to assist in carrying out the required calculations.

  17. Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling

    OpenAIRE

    Motorin, Yuri; Burhenne, Jürgen; Teimer, Roman; Koynov, Kaloian; Willnow, Sophie; Weinhold, Elmar; Helm, Mark

    2010-01-01

    This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNAPhe. LC/MS a...

  18. Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For the temporally and spatially regulated expression of the barnase gene in plant,two kinds of plasmids with cre gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure.The transgenic plants were conformed by PCR analysis.The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome.Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.

  19. Identification of RNA sequences and structures involved in site-specific cleavage of IGF-II mRNAs.

    OpenAIRE

    van Dijk, E L; Sussenbach, J S; Holthuizen, P E

    1998-01-01

    Insulin-like growth factor-II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region (UTR), rendering an unstable 5' cleavage product containing the coding region and a very stable 3' cleavage product of 1.8 kb consisting of the 3'-UTR sequence and the poly(A) tail. Previously, it was established that two widely separated elements in the 3'-UTR (elements I and II), that can form a duplex structure, are necessary and sufficient for cleavage. To furth...

  20. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    International Nuclear Information System (INIS)

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL−1 of MBD2 with a linear range of 0.2–300 ng mL−1. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL−1 (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL−1 and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no requirement of bisulfite

  1. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  2. Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system.

    Science.gov (United States)

    Karimova, Madina; Abi-Ghanem, Josephine; Berger, Nicolas; Surendranath, Vineeth; Pisabarro, Maria Teresa; Buchholz, Frank

    2013-01-01

    Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo. However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/vox. Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/vox is a novel powerful and safe instrument in the 'genetic toolbox' that can be used alone or in combination with other SSRs in heterologous hosts. PMID:23143104

  3. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

    Science.gov (United States)

    Nienberg, Christian; Retterath, Anika; Becher, Kira-Sophie; Saenger, Thorsten; Mootz, Henning D; Jose, Joachim

    2016-06-27

    Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

  4. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications

    Directory of Open Access Journals (Sweden)

    Christian Nienberg

    2016-06-01

    Full Text Available Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

  5. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications †

    Science.gov (United States)

    Nienberg, Christian; Retterath, Anika; Becher, Kira-Sophie; Saenger, Thorsten; Mootz, Henning D.; Jose, Joachim

    2016-01-01

    Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay. PMID:27355959

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

    Science.gov (United States)

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

    2015-03-01

    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. PMID:25654326

  7. Site-specific proteolysis of the transcriptional coactivator HCF-1 can regulate its interaction with protein cofactors.

    Science.gov (United States)

    Vogel, Jodi L; Kristie, Thomas M

    2006-05-01

    Limited proteolytic processing is an important transcriptional regulatory mechanism. In various contexts, proteolysis controls the cytoplasmic-to-nuclear transport of important transcription factors or removes domains to produce factors with altered activities. The transcriptional coactivator host cell factor-1 (HCF-1) is proteolytically processed within a unique domain consisting of 20-aa reiterations. Site-specific cleavage within one or more repeats generates a family of amino- and carboxyl-terminal subunits that remain tightly associated. However, the consequences of HCF-1 processing have been undefined. In this study, it was determined that the HCF-1-processing domain interacts with several proteins including the transcriptional coactivator/corepressor four-and-a-half LIM domain-2 (FHL2). Analysis of this interaction has uncovered specificity with both sequence and context determinants within the reiterations of this processing domain. In cells, FHL2 interacts exclusively with the nonprocessed coactivator and costimulates transcription of an HCF-1-dependent target gene. The functional interaction of HCF-1 with FHL2 supports a model in which site-specific proteolysis regulates the interaction of HCF-1 with protein partners and thus can modulate the activity of this coactivator. This paradigm expands the biological significance of limited proteolytic processing as a regulatory mechanism in gene transcription.

  8. Too packed to change: side-chain packing and site-specific substitution rates in protein evolution

    Directory of Open Access Journals (Sweden)

    María Laura Marcos

    2015-04-01

    Full Text Available In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN, the sum of inverse square distances between a site’s Cα and the Cα of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein’s active conformation. While WCN is a measure of Cα packing, mutations replace side chains. Here, we consider whether a site’s evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA. We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation. Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.

  9. Too packed to change: side-chain packing and site-specific substitution rates in protein evolution.

    Science.gov (United States)

    Marcos, María Laura; Echave, Julian

    2015-01-01

    In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN), the sum of inverse square distances between a site's C α and the C α of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein's active conformation. While WCN is a measure of C α packing, mutations replace side chains. Here, we consider whether a site's evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA). We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation). Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.

  10. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    Science.gov (United States)

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis. PMID:10880566

  11. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    Science.gov (United States)

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  12. Shaping the lens of the atom probe: Fabrication of site specific, oriented specimens and application to grain boundary analysis

    International Nuclear Information System (INIS)

    The random sampling provided by classical atom probe sample preparation methods is one of the major factors limiting the types of problems that can be addressed using this powerful technique. A focused ion beam enables not only site-specific preparation, but can also be used to give the specimen, which acts as the lens in an atom probe experiment, a specific shape. In this paper we present a technique that uses low accelerating voltages (10 and 5 kV) in the focused ion beam (FIB) to reproducibly produce specimens with selected grain boundaries <100 nm from the tip at any desired orientation. These tips have a high rate of successfully running in the atom probe and no Ga contamination within the region of interest. This technique is applied to the analysis of grain boundaries in a high purity iron wire and a strip-cast steel. Lattice resolution is achieved around the boundary in certain areas. Reconstruction of these datasets reveals the distribution of light and heavy elements around the boundary. Issues surrounding the uneven distribution of certain solute elements as a result of field-induced diffusion are discussed. -- Research highlights: → Damage free site specific samples can be made using FIB. → Crystallographic relationships across grain boundaries can be obtained from field desorption patterns. → Lattice resolution can be achieved at grain boundaries for matrix atoms. → Field evaporation artifacts are observed at grain boundaries for solute atoms.

  13. Long-range chromosome organization in E. coli: a site-specific system isolates the Ter macrodomain.

    Directory of Open Access Journals (Sweden)

    Axel Thiel

    Full Text Available The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery.

  14. Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

    Science.gov (United States)

    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.

  15. Derivation of site-specific relationships between hydraulic parameters and p-wave velocities based on hydraulic and seismic tomography

    Energy Technology Data Exchange (ETDEWEB)

    Brauchler, R.; Doetsch, J.; Dietrich, P.; Sauter, M.

    2012-01-10

    In this study, hydraulic and seismic tomographic measurements were used to derive a site-specific relationship between the geophysical parameter p-wave velocity and the hydraulic parameters, diffusivity and specific storage. Our field study includes diffusivity tomograms derived from hydraulic travel time tomography, specific storage tomograms, derived from hydraulic attenuation tomography, and p-wave velocity tomograms, derived from seismic tomography. The tomographic inversion was performed in all three cases with the SIRT (Simultaneous Iterative Reconstruction Technique) algorithm, using a ray tracing technique with curved trajectories. The experimental set-up was designed such that the p-wave velocity tomogram overlaps the hydraulic tomograms by half. The experiments were performed at a wellcharacterized sand and gravel aquifer, located in the Leine River valley near Göttingen, Germany. Access to the shallow subsurface was provided by direct-push technology. The high spatial resolution of hydraulic and seismic tomography was exploited to derive representative site-specific relationships between the hydraulic and geophysical parameters, based on the area where geophysical and hydraulic tests were performed. The transformation of the p-wave velocities into hydraulic properties was undertaken using a k-means cluster analysis. Results demonstrate that the combination of hydraulic and geophysical tomographic data is a promising approach to improve hydrogeophysical site characterization.

  16. Geothermal investment analysis with site-specific applications to Roosevelt Hot Springs and Cove Fort-Sulphurdale, Utah

    Energy Technology Data Exchange (ETDEWEB)

    Cassel, T.A.V.; Edelstein, R.H.; Blair, P.D.

    1978-12-01

    The analysis and modeling of investment behavior in the development of hydrothermal electric power facilities are reported. This investment behavior reflects a degree of sensitivity to public policy alternatives concerning taxation and regulation of the resource and its related energy conversion facilities. The objective of the current research is to provide a realistic and theoretically sound means for estimating the impacts of such public policy alternatives. A stochastic simulation model was developed which offers an efficient means for site-specific investment analysis of private sector firms and investors. The results of the first year of work are discussed including the identification, analysis, quantification and modeling of: a decision tree reflecting the sequence of procedures, timing and stochastic elements of hydrothermal resource development projects; investment requirements, expenses and revenues incurred in the exploration, development and utilization of hydrothermal resources for electric power generation; and multiattribute investment decision criteria of the several types of firms in the geothermal industry. An application of the investment model to specific resource sites in the state of Utah is also described. Site specific data for the Known Geothermal Resource Areas of Roosevelt Hot Springs and Cove Fort-Sulphurdale are given together with hypothesized generation capacity growth rates.

  17. Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

    Science.gov (United States)

    Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

    2008-04-01

    The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

  18. A novel approach for site-specific atom probe specimen preparation by focused ion beam and transmission electron backscatter diffraction

    International Nuclear Information System (INIS)

    Atom probe tomography (APT) is a suitable technique for chemical analyses with almost atomic resolution. However, the time-consuming site-specific specimen preparation can be improved. Recently, transmission electron backscatter diffraction (t-EBSD) has been established for high resolution crystallographic analyses of thin foils. In this paper we present the first successful application of a combined focused ion beam (FIB)/t-EBSD preparation of site-specific APT specimens using the example of grain boundary segregation in technically pure molybdenum. It will be shown that the preparation of a grain boundary can be substantially accelerated by t-EBSD analyses in-between the annular milling FIB procedure in the same microscope. With this combined method, a grain boundary can easily be recognized and positioned in the first 220 nm of an APT sample much faster than e.g. with complementary investigations in a transmission electron microscope. Even more, the high resolution technique of t-EBSD gives the opportunity to get crystallographic information of the mapped area and, therefore, an analysis of the grain boundary character to support the interpretation of the APT data files. To optimize this newly developed technique for the application on needle-shaped APT specimens, a parameter study on enhanced background correction, acceleration voltage, and tilt angle was carried out. An acceleration voltage of 30 kV at specimen surface tilt angles between −45° and −35° from horizontal plane leads to the best results. Even for molybdenum the observation of crystal orientation data up to about 200 nm specimen thickness is possible. - Highlights: • We developed a new site-specific APT specimen preparation method by FIB and t-EBSD. • A grain boundary was positioned in the first 220 nm of the APT tip by the FIB/t-EBSD method. • Crystallographic information of the mapped area can be quickly obtained. • An acceleration voltage of 30 kV at specimen surface tilt angles

  19. Binding of cationic surfactants to humic substances

    NARCIS (Netherlands)

    Ishiguro, M.; Tan, W.; Koopal, L.K.

    2007-01-01

    Commercial surfactants are introduced into the environment either through waste products or site-specific contamination. The amphiphilic nature of both surfactants and humic substances (HS) leads to their mutual attraction especially when surfactant and HS are oppositely charged. Binding of the cati

  20. Tracking Site-specific C-C Coupling of Formaldehyde Molecules on Rutile TiO2(110)

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Ke; Xia, Yaobiao; Tang, Miru; Wang, Zhitao; Jan, Bryan; Lyubinetsky, Igor; Ge, Qingfeng; Dohnalek, Zdenek; Park, Kenneth T.; Zhang, Zhenrong

    2015-06-25

    Direct imaging of site-specific reactions of individual mole-cules as a function of temperature is a long-sought goal in molecular science. Here, we report the direct visualization of molecular coupling of formaldehyde on reduced rutile TiO2(110) surfaces as we track the same set of molecules when the temperature is increased from 75 to 170 K using scanning tunneling microscope (STM). Our recent study showed that formaldehyde preferably adsorbs on bridging-bonded oxygen (Ob) vacancy (VO) defect site. Herein, images from the same area as the temperature is increased show that VO-bound formaldehyde couples with Ti-bound formaldehyde forming a diolate intermediate. Exposure of formaldehyde at room temperature leads to diolate as the majority species on the surface and no VO-bound formaldehyde is observed. The diolate species are the key reaction intermediates in the formation of ethylene reported in previous ensemble-averaged studies.

  1. Development of a Site-specific Standard for Selenium in Open Waters of Great Salt Lake, Utah

    Science.gov (United States)

    Moellmer, W. O.; Miller, T.; Ohlendorf, H.; Denbleyker, J.

    2006-12-01

    The Great Salt Lake (GSL) is a unique terminal lake located adjacent to a rapidly growing metropolitan area in the western United States. The open water of the GSL is protected for its current beneficial uses through the application of a narrative criteria clause in the state water quality standards. The Utah Department of Environmental Quality (DEQ) initiated a process in 2004 to develop a site-specific numeric water quality standard for selenium for the open waters of the GSL to balance protection of the GSL's unique ecology and beneficial uses with burgeoning development. The process the DEQ initiated included the formation of a Great Salt Lake Water Quality Steering Committee and a Science Panel to identify the studies required, manage the studies and finally recommend a site-specific standard. Great Salt Lake Water Quality Steering Committee. The DEQ established the GSL Water Quality Steering Committee (Steering Committee) to provide a forum for stakeholders to assist in guiding the process of developing numeric standards for the lake. This group consists of federal and state regulatory agencies, other public entities, conservation organizations, recreation groups, and industrial users of the lake. Great Salt Lake Science Panel. The DEQ established the GSL Science Panel (Science Panel) to advise the DEQ and Steering Committee and provide overall technical direction and review for the program. The Science Panel is composed of 9 members representing federal and state regulatory agencies, industry and academia. The purpose of the panel is to identify data gaps in the literature, design and oversee scientific investigations to fill critical data gaps, and finally recommend a numeric water quality standard to the Steering Committee. Studies Currently Underway. A partnership of researchers—including local and national experts from education and industry—are collaborating with the DEQ, the Steering Committee, and the Science Panel to complete the studies required

  2. Human health and ecological risk assessment of soil-borne arsenic and lead: A site-specific risk assessment

    Energy Technology Data Exchange (ETDEWEB)

    Roy, M.; Epp, G.A.; Beukema, P. [Proctor and Redfern Ltd., Don Mills, Ontario (Canada); Nieboer, E. [McMaster Univ., Hamilton, Ontario (Canada)

    1997-12-31

    Screening level site specific human health and ecological risk assessments (ERA) were conducted at a historical (1908--1921) smelting and refining site in the Niagara Region, Ontario in accordance with the recently released provincial and federal risk assessment guidelines. The purpose of the assessment was to evaluate the risk associated with elevated levels of arsenic and lead in surface soils, and to assess alternative remediation options, prior to property transfer. Future intended land use will be parkland and for the site to remain forested. The identification of potential receptors, exposure pathways, and end-points was conducted at the biological community-level. The ERA involved a toxic cue inventory of the core smelting and refining site, adjacent lands and a reference site. Development of remediation options was based on hazard assessment and the prediction of risks associated with arsenic contamination. An evaluation of remediation options and the selection of a preferred option are discussed.

  3. The joint industry development of a recommended practice for the site specific assessment of mobile jackup units

    International Nuclear Information System (INIS)

    The mobile self-elevating (drilling) unit, or jack-up, has been central to the exploration and development of offshore oil and gas reserves. In recent years there has been an increased desire to benefit from the potential economics of using these units in longer term production roles either in association with a fixed platform or as a stand-alone facility. However, until recently there had been no concerted effort to develop a consistent and commonly accepted standard for general industry use in site specific assessment of jack-ups. This paper describes the background and work carried out to develop such a standard. The project is nearing completion and has resulted in the publication of a Guideline document. A Recommended Practice and associated Commentary are (at the time of writing) in the final stage of drafting for release to the project sponsors for trial and comment prior to issuing the first Edition early in 1994

  4. A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

    DEFF Research Database (Denmark)

    Goth, Christoffer K; Halim, Adnan; Khetarpal, Sumeet A;

    2015-01-01

    -glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated...... by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme...... and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell...

  5. Development of Site-Specific Soil Design Basis Earthquake (DBE) Parameters for the Integrated Waste Treatment Unit (IWTU)

    Energy Technology Data Exchange (ETDEWEB)

    Payne, Suzette

    2008-08-01

    Horizontal and vertical PC 3 (2,500 yr) Soil Design Basis Earthquake (DBE) 5% damped spectra, corresponding time histories, and strain-compatible soil properties were developed for the Integrated Waste Treatment Unit (IWTU). The IWTU is located at the Idaho Nuclear Technology and Engineering Center (INTEC) at the Idaho National Laboratory (INL). Mean and 84th percentile horizontal DBE spectra derived from site-specific site response analyses were evaluated for the IWTU. The horizontal and vertical PC 3 (2,500 yr) Soil DBE 5% damped spectra at the 84th percentile were selected for Soil Structure Interaction (SSI) analyses at IWTU. The site response analyses were performed consistent with applicable Department of Energy (DOE) Standards, recommended guidance of the Nuclear Regulatory Commission (NRC), American Society of Civil Engineers (ASCE) Standards, and recommendations of the Blue Ribbon Panel (BRP) and Defense Nuclear Facilities Safety Board (DNFSB).

  6. CoPc and CoPcF16 on gold: Site-specific charge-transfer processes

    Directory of Open Access Journals (Sweden)

    Fotini Petraki

    2014-04-01

    Full Text Available Interface properties of cobalt(II phthalocyanine (CoPc and cobalt(II hexadecafluoro-phthalocyanine (CoPcF16 to gold are investigated by photo-excited electron spectroscopies (X-ray photoemission spectroscopy (XPS, ultraviolet photoemission spectroscopy (UPS and X-ray excited Auger electron spectroscopy (XAES. It is shown that a bidirectional charge transfer determines the interface energetics for CoPc and CoPcF16 on Au. Combined XPS and XAES measurements allow for the separation of chemical shifts based on different local charges at the considered atom caused by polarization effects. This facilitates a detailed discussion of energetic shifts of core level spectra. The data allow the discussion of site-specific charge-transfer processes.

  7. Dry Deposition Velocity Estimation for the Savannah River Site: Part 2 -- Parametric and Site-Specific Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Napier, Bruce A.; Rishel, Jeremy P.; Cook, Kary M.

    2013-09-12

    Values for the dry deposition velocity of airborne particles were estimated with the GENII Version 2.10.1 computer code for the Savannah River site using assumptions about surface roughness parameters and particle size and density. Use of the GENII code is recommended by the U.S. Department of Energy for this purpose. Meteorological conditions evaluated include atmospheric stability classes D, E, and F and wind speeds of 0.5, 1.0, 1.5, and 2.0 m/s. Local surface roughness values ranging from 0.03 to 2 meters were evaluated. Particles with mass mean diameters of 1, 5, and 10 microns and densities of 1, 3, 4, and 5 g/cm3 were evaluated. Site specific meteorology was used to predict deposition velocity for Savannah River conditions for a range of distances from 670 to 11,500 meters.

  8. Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector.

    Science.gov (United States)

    Van Mellaert, L; Mei, L; Lammertyn, E; Schacht, S; Anné, J

    1998-12-01

    The temperate bacteriophage VWB integrates into the chromosome of Streptomyces venezuelae ETH14630 via site-specific integration. Following recombination of the VWB attP region with the chromosomal attB sequence, the host-phage junctions attL and attR are formed. Nucleotide sequence analysis of attP, attB, attL and attR revealed a 45 bp common core sequence. In attB this 45 bp sequence consists of the 3' end of a putative tRNA Arg(AGG) gene with a 3'-terminal CCA sequence which is typical for prokaryotic tRNAs. Phage DNA integration restores the putative tRNA Arg(AGG) gene in attL. However, following recombination the CCA sequence is missing as is the case for most Streptomyces tRNA genes described so far. Adjacent to VWB attP, an ORF encoding a 427 aa protein was detected. The C-terminal region of this protein shows high similarity to the conserved C-terminal domain of site-specific recombinases belonging to the integrase family. To prove the functionality of this putative integrase gene (int), an integrative vector pKT02 was constructed. This vector consists of a 2.3 kb HindIII-SphI restriction fragment of VWB DNA containing attP and int cloned in a non-replicative Escherichia coli vector carrying a thiostrepton-resistance (tsr) gene. Integration of pKT02 was obtained after transformation of Streptomyces venezuelae ETH14630 and Streptomyces lividans TK24 protoplasts. This vector will thus be useful for a number of additional Streptomyces species in which a suitable tRNA gene can be functional as integration site.

  9. Remote Sensing and Geospatial Technological Applications for Site-specific Management of Fruit and Nut Crops: A Review

    Directory of Open Access Journals (Sweden)

    Joel O. Paz

    2010-08-01

    Full Text Available Site-specific crop management (SSCM is one facet of precision agriculture which is helping increase production with minimal input. It has enhanced the cost-benefit scenario in crop production. Even though the SSCM is very widely used in row crop agriculture like corn, wheat, rice, soybean, etc. it has very little application in cash crops like fruit and nut. The main goal of this review paper was to conduct a comprehensive review of advanced technologies, including geospatial technologies, used in site-specific management of fruit and nut crops. The review explores various remote sensing data from different platforms like satellite, LIDAR, aerial, and field imaging. The study analyzes the use of satellite sensors, such as Quickbird, Landsat, SPOT, and IRS imagery as well as hyperspectral narrow-band remote sensing data in study of fruit and nut crops in blueberry, citrus, peach, apple, etc. The study also explores other geospatial technologies such as GPS, GIS spatial modeling, advanced image processing techniques, and information technology for suitability study, orchard delineation, and classification accuracy assessment. The study also provides an example of a geospatial model developed in ArcGIS ModelBuilder to automate the blueberry production suitability analysis. The GIS spatial model is developed using various crop characteristics such as chilling hours, soil permeability, drainage, and pH, and land cover to determine the best sites for growing blueberry in Georgia, U.S. The study also provides a list of spectral reflectance curves developed for some fruit and nut crops, blueberry, crowberry, redblush citrus, orange, prickly pear, and peach. The study also explains these curves in detail to help researchers choose the image platform, sensor, and spectrum wavelength for various fruit and nut crops SSCM.

  10. Multifactorial, site-specific recurrence models after radical cystectomy for urothelial carcinoma: external validation in a cohort of Korean patients.

    Directory of Open Access Journals (Sweden)

    Hyung Suk Kim

    Full Text Available The aim of this study was to evaluate the accuracy of site-specific recurrence models after radical cystectomy in the Korean population.We conducted a review of an electronic medical record of 572 patients who underwent radical cystectomy for urothelial carcinoma of the bladder. Primary end point was the site-specific recurrence after radical cystectomy.The median follow-up in the validation cohort was 42.3 months (interquartile range: 23.0-89.3 months. During the follow-up period, there were 165 patients (28.8%, 85 (14.9%, 31 (5.4%, and 78 (13.6% who recurred in abdomen/pelvis, thoracic region, upper urinary tract, and bone, respectively. The c-indices of abdomen/pelvis, thoracic region, upper urinary tract, and bone models 3 years after radical cystectomy were 0.69 (95% confidence interval [CI], 0.65-0.73, 0.69 (95% CI, 0.64-0.75, 0.61 (95% CI, 0.52-0.69, and 0.65 (95% CI, 0.59-0.71, respectively. Kaplan-Meier curves demonstrated that models discriminated well and log-rank test were all highly significant (all p<0.001, except upper urinary tract model (p = 0.366. Decision curve analysis revealed that the use of prediction models for abdomen/pelvis, thoracic region, and bone recurrence was associated with net benefit gains relative to the treat-all strategy, but not the model for upper urinary tract recurrence.Abdomen/pelvis, thoracic region, and bone models demonstrate moderate discrimination, adequate calibration, and meaningful net benefit gains, whereas upper urinary tract model does not seem applicable to patients from Asia because it has suboptimal accuracy.

  11. Evaluation of site-specific factors influencing heavy metal contents in the topsoil of vegetated infiltration swales.

    Science.gov (United States)

    Horstmeyer, Nils; Huber, Maximilian; Drewes, Jörg E; Helmreich, Brigitte

    2016-08-01

    Stormwater runoff of traffic areas is usually polluted by organic and inorganic substances and must be treated prior to discharge into groundwater. One widely used treatment method is infiltrating the runoff over the topsoil of vegetated swales. The aim of this study was to evaluate the factors influencing the heavy metal contents in such topsoil layers of vegetated infiltration swales near highways, roads, and parking lots. In total, 262 topsoil samples were taken from 35 sampling sites, which varied in age, traffic volume, road design, driving style, and site-specific conditions. In the evaluation of all soil samples, the median heavy metal values of cadmium, chromium, copper, lead, and zinc were yielding 0.36 (mean: 1.21) mg/kg DM, 37.0 (mean: 44.5) mg/kg DM, 28.0 (mean: 61.5) mg/kg DM, 27.0 (mean: 71.9) mg/kg DM, and 120 (mean: 257) mg/kg DM, respectively. The main purpose was to evaluate the site-specific data (i.e., surrounding land use characteristics, traffic area site data, and operational characteristics). In general, heavy metal contents increased with increasing traffic volumes. However, other factors also had a notable impact. Factors such as road design (e.g., curves, crossings, and roundabouts) and grade of congestion significantly influenced the heavy metal contents. High heavy metal contents were detected for stop-and-go areas, roundabouts, crossings, and sites with traffic lights, signs, and guardrails. Findings of this study can be used to identify highly polluted traffic areas and to verify or improve standards regarding the treatment of runoff from traffic areas. PMID:27093119

  12. Evaluation of site-specific factors influencing heavy metal contents in the topsoil of vegetated infiltration swales.

    Science.gov (United States)

    Horstmeyer, Nils; Huber, Maximilian; Drewes, Jörg E; Helmreich, Brigitte

    2016-08-01

    Stormwater runoff of traffic areas is usually polluted by organic and inorganic substances and must be treated prior to discharge into groundwater. One widely used treatment method is infiltrating the runoff over the topsoil of vegetated swales. The aim of this study was to evaluate the factors influencing the heavy metal contents in such topsoil layers of vegetated infiltration swales near highways, roads, and parking lots. In total, 262 topsoil samples were taken from 35 sampling sites, which varied in age, traffic volume, road design, driving style, and site-specific conditions. In the evaluation of all soil samples, the median heavy metal values of cadmium, chromium, copper, lead, and zinc were yielding 0.36 (mean: 1.21) mg/kg DM, 37.0 (mean: 44.5) mg/kg DM, 28.0 (mean: 61.5) mg/kg DM, 27.0 (mean: 71.9) mg/kg DM, and 120 (mean: 257) mg/kg DM, respectively. The main purpose was to evaluate the site-specific data (i.e., surrounding land use characteristics, traffic area site data, and operational characteristics). In general, heavy metal contents increased with increasing traffic volumes. However, other factors also had a notable impact. Factors such as road design (e.g., curves, crossings, and roundabouts) and grade of congestion significantly influenced the heavy metal contents. High heavy metal contents were detected for stop-and-go areas, roundabouts, crossings, and sites with traffic lights, signs, and guardrails. Findings of this study can be used to identify highly polluted traffic areas and to verify or improve standards regarding the treatment of runoff from traffic areas.

  13. Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo.

    Science.gov (United States)

    Yang, Byungseop; Lim, Sung In; Kim, Jong Chul; Tae, Giyoong; Kwon, Inchan

    2016-05-01

    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. PMID:27050863

  14. Analysis of High Frequency Site-Specific Nitrogen and Oxygen Isotopic Composition of Atmospheric Nitrous Oxide at Mace Head, Ireland

    Science.gov (United States)

    McClellan, M. J.; Harris, E. J.; Olszewski, W.; Ono, S.; Prinn, R. G.

    2014-12-01

    Atmospheric nitrous oxide (N2O) significantly impacts Earth's climate due to its dual role as an inert potent greenhouse gas in the troposphere and as a reactive source of ozone-destroying nitrogen oxides in the stratosphere. However, there remain significant uncertainties in the global budget of this gas. The marked spatial divide in its reactivity means that all stages in the N2O life cycle—emission, transport, and destruction—must be examined to understand the overall effect of N2O on climate. Source and sink processes of N2O lead to varying concentrations of N2O isotopologues (14N14N16O, 14N15N16O, 15N14N16O, and 14N14N18O being measured) due to preferential isotopic production and elimination in different environments. Estimation of source and sink fluxes can be improved by combining isotopically resolved N2O observations with simulations using a chemical transport model with reanalysis meteorology and treatments of isotopic signatures of specific surface sources and stratospheric intrusions. We present the first few months of site-specific nitrogen and oxygen isotopic composition data from the Stheno-TILDAS instrument (Harris et al, 2013) at Mace Head, Ireland and compare these to results from MOZART-4 (Model for Ozone and Related Chemical Tracers, version 4) chemical transport model runs including N2O isotopic fractionation processes and reanalysis meterological fields (NCEP/NCAR, MERRA, and GEOS-5). This study forms the basis for future inverse modeling experiments that will improve the accuracy of isotopically differentiated N2O emission and loss estimates. Ref: Harris, E., D. Nelson, W. Olszewski, M. Zahniser, K. Potter, B. McManus, A. Whitehill, R. Prinn, and S. Ono, Development of a spectroscopic technique for continuous online monitoring of oxygen and site-specific nitrogen isotopic composition of atmospheric nitrous oxide, Analytical Chemistry, 2013; DOI: 10.1021/ac403606u.

  15. Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

    Science.gov (United States)

    Simard, Bryan; Mironov, Gleb G; Tomanek, Boguslaw; van Veggel, Frank C J M; Abulrob, Abedelnasser

    2016-06-01

    Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27282138

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

    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.

  18. Report of the Department of Energy (DOE) Office of Energy Research Review Committee on the site-specific conceptual design of the Superconducting Super Collider

    International Nuclear Information System (INIS)

    After it was established in early 1989, the Superconducting Super Collider Laboratory (SSCL) began to prepare a detailed site-specific SSC conceptual design, including cost and schedule estimates. As detailed in the SSC Site-Specific Conceptual Design Report (SCDR), this design builds upon the design in the March 1986 SSC Conceptual Design Report (CDR) and takes into account characteristics of the SSC site, results of continuing magnet R ampersand D, and advances in accelerator design

  19. Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.

    OpenAIRE

    Collier, D A; Mergny, J L; Thuong, N T; Helene, C

    1991-01-01

    Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs...

  20. Site-specific reaction rate constant measurements for various secondary and tertiary H-abstraction by OH radicals

    KAUST Repository

    Badra, Jihad

    2015-02-01

    Reaction rate constants for nine site-specific hydrogen atom (H) abstraction by hydroxyl radicals (OH) have been determined using experimental measurements of the rate constants of Alkane+OH→Products reactions. Seven secondary (S 20, S 21, S 22, S 30, S 31, S 32, and S 33) and two tertiary (T 100 and T 101) site-specific rate constants, where the subscripts refer to the number of carbon atoms (C) connected to the next-nearest-neighbor (N-N-N) C atom, were obtained for a wide temperature range (250-1450K). This was done by measuring the reaction rate constants for H abstraction by OH from a series of carefully selected large branched alkanes. The rate constant of OH with four different alkanes, namely 2,2-dimethyl-pentane, 2,4-dimethyl-pentane, 2,2,4-trimethyl-pentane (iso-octane), and 2,2,4,4-tetramethyl-pentane were measured at high temperatures (822-1367K) using a shock tube and OH absorption diagnostic. Hydroxyl radicals were detected using the narrow-line-width ring-dye laser absorption of the R1(5) transition of OH spectrum near 306.69nm.Previous low-temperature rate constant measurements are added to the current data to generate three-parameter rate expressions that successfully represent the available direct measurements over a wide temperature range (250-1450. K). Similarly, literature values of the low-temperature rate constants for the reaction of OH with seven normal and branched alkanes are combined with the recently measured high-temperature rate constants from our group [1]. Subsequent to that, site-specific rate constants for abstractions from various types of secondary and tertiary H atoms by OH radicals are derived and have the following modified Arrhenius expressions:. S20=8.49×10-17T1.52exp(73.4K/T)cm3molecule-1s-1(250-1450K) S21=1.07×10-15T1.07exp(208.3K/T)cm3molecule-1s-1(296-1440K) S22=2.88×10-13T0.41exp(-291.5K/T)cm3molecule-1s-1(272-1311K) S30=3.35×10-18T1.97exp(323.1K/T)cm3molecule-1s-1(250-1366K) S31=1.60×10-18T2.0exp(500.0K/T)cm3