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Sample records for linker sequences rich

  1. Description of a cellulose-binding domain and a linker sequence from Aspergillus fungi

    NARCIS (Netherlands)

    Quentin, M; Ebbelaar, M; Derksen, J; Mariani, C; van der Valk, H

    A family I cellulose-binding domain (CBD) and a serine- and threonine-rich linker peptide were cloned from the fungi Aspergillus japonicus and Aspergillus aculeatus. A glutathione S-transferase (GST) fusion protein comprising GST and a peptide linker with the CBD fused to its C-terminus, was

  2. Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation.

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    Deanne W Sammond

    Full Text Available Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs to carbohydrate-binding modules (CBMs. Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests

  3. Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase.

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    Meiselbach, Heike; Sticht, Heinrich

    2011-08-01

    The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family tyrosine kinases, which are found in many signaling pathways, differ in their physiological function despite their high overall structural similarity. In this context, the differences in the SH3-SH2 domain linkers might play a role for differential regulation, but the structural consequences of linker sequence remain poorly understood. We have therefore performed comparative molecular dynamics simulations of wildtype Hck and of a mutant Hck in which the SH3-SH2 domain linker is replaced by the corresponding sequence from the homologous kinase Lck. These simulations reveal that linker replacement not only affects the orientation of the SH3 domain itself, but also leads to an alternative conformation of the activation segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a remote effect on the active site geometry and might therefore play a role in modulating the structure of the inactive kinase or in fine-tuning the activation process itself.

  4. Bioinformatic Analysis Reveals Conservation of Intrinsic Disorder in the Linker Sequences of Prokaryotic Dual-family Immunophilin Chaperones.

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    Barik, Sailen

    2018-01-01

    The two classical immunophilin families, found essentially in all living cells, are: cyclophilin (CYN) and FK506-binding protein (FKBP). We previously reported a novel class of immunophilins that are natural chimera of these two, which we named dual-family immunophilin (DFI). The DFIs were found in either of two conformations: CYN-linker-FKBP (CFBP) or FKBP-3TPR-CYN (FCBP). While the 3TPR domain can serve as a flexible linker between the FKBP and CYN modules in the FCBP-type DFI, the linker sequences in the CFBP-type DFIs are relatively short, diverse in sequence, and contain no discernible motif or signature. Here, I present several lines of computational evidence that, regardless of their primary structure, these CFBP linkers are intrinsically disordered. This report provides the first molecular foundation for the model that the CFBP linker acts as an unstructured, flexible loop, allowing the two flanking chaperone modules function independently while linked in cis , likely to assist in the folding of multisubunit client complexes.

  5. Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

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    Sauer, J; Christensen, T; Frandsen, T P; Mirgorodskaya, E; McGuire, K A; Driguez, H; Roepstorff, P; Sigurskjold, B W; Svensson, B

    2001-08-07

    Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

  6. Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA

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    Shinjiro Sawada

    2017-10-01

    Full Text Available DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

  7. Optimization of the Alkyl Linker of TO Base Surrogate in Triplex-Forming PNA for Enhanced Binding to Double-Stranded RNA.

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    Sato, Takaya; Sato, Yusuke; Nishizawa, Seiichi

    2017-03-23

    A series of triplex-forming peptide nucleic acid (TFP) probes carrying a thiazole orange (TO) base surrogate through an alkyl linker was synthesized, and the interactions between these so-called tFIT probes and purine-rich sequences within double-stranded RNA (dsRNA) were examined. We found that the TO base surrogate linker significantly affected both the binding affinity and the fluorescence response upon triplex formation with the target dsRNA. Among the probes examined, the TO base surrogate connected through the propyl linker in the tFIT probes increased the binding affinity by a factor of ten while maintaining its function as the fluorescent universal base. Isothermal titration calorimetry experiments revealed that the increased binding affinity resulted from the gain in the binding enthalpy, which could be explained by the enhanced π-stacking interaction between the TO base surrogate and the dsRNA part of the triplex. We expect that these results will provide a molecular basis for designing strong binding tFIT probes for fluorescence sensing of various kinds of purine-rich dsRNAs sequences including those carrying a pyrimidine-purine inversion. The obtained data also offers a new insight into further development of the universal bases incorporated in TFP. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS)

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    Iacobucci, Claudio; Hage, Christoph; Schäfer, Mathias; Sinz, Andrea

    2017-10-01

    The chemical cross-linking/mass spectrometry (MS) approach is a growing research field in structural proteomics that allows gaining insights into protein conformations. It relies on creating distance constraints between cross-linked amino acid side chains that can further be used to derive protein structures. Currently, the most urgent task for designing novel cross-linking principles is an unambiguous and automated assignment of the created cross-linked products. Here, we introduce the homobifunctional, amine-reactive, and water soluble cross-linker azobisimidoester (ABI) as a prototype of a novel class of cross-linkers. The ABI-linker possesses an innovative modular scaffold combining the benefits of collisional activation lability with open shell chemistry. This MS-cleavable cross-linker can be efficiently operated via free radical initiated peptide sequencing (FRIPS) in positive ionization mode. Our proof-of-principle study challenges the gas phase behavior of the ABI-linker for the three amino acids, lysine, leucine, and isoleucine, as well as the model peptide thymopentin. The isomeric amino acids leucine and isoleucine could be discriminated by their characteristic side chain fragments. Collisional activation experiments were conducted via positive electrospray ionization (ESI) on two Orbitrap mass spectrometers. The ABI-mediated formation of odd electron product ions in MS/MS and MS3 experiments was evaluated and compared with a previously described azo-based cross-linker. All cross-linked products were amenable to automated analysis by the MeroX software, underlining the future potential of the ABI-linker for structural proteomics studies. [Figure not available: see fulltext.

  9. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

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    Sheila M Reynolds

    2010-07-01

    Full Text Available DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the

  10. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

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    Reynolds, Sheila M; Bilmes, Jeff A; Noble, William Stafford

    2010-07-08

    DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the

  11. Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators

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    Teresa Milano

    2016-12-01

    Full Text Available Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro–Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties.

  12. Learning a Weighted Sequence Model of the Nucleosome Core and Linker Yields More Accurate Predictions in Saccharomyces cerevisiae and Homo sapiens

    Science.gov (United States)

    Reynolds, Sheila M.; Bilmes, Jeff A.; Noble, William Stafford

    2010-01-01

    DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence—301 base pairs, centered at the position to be scored—with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the

  13. SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

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    Register, A C; Leonard, Stephen E; Maly, Dustin J

    2014-11-11

    Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

  14. A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor.

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    Vishwanatha, Kurutihalli; Bäck, Nils; Mains, Richard E; Eipper, Betty A

    2014-05-02

    Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

  15. Novel Concepts of MS-Cleavable Cross-linkers for Improved Peptide Structure Analysis

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    Hage, Christoph; Falvo, Francesco; Schäfer, Mathias; Sinz, Andrea

    2017-10-01

    The chemical cross-linking/mass spectrometry (MS) approach is gaining increasing importance as an alternative method for studying protein conformation and for deciphering protein interaction networks. This study is part of our ongoing efforts to develop innovative cross-linking principles for a facile and efficient assignment of cross-linked products. We evaluate two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for automated analysis of cross-linked products. We introduce the bromine phenylurea (BrPU) linker that possesses a unique structure yielding a distinctive fragmentation pattern on collisional activation. Moreover, BrPU delivers the characteristic bromine isotope pattern and mass defect for all cross-linker-decorated fragments. We compare the fragmentation behavior of the BrPU linker with that of our previously described MS-cleavable TEMPO-Bz linker (which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy moiety connected to a benzyl group) that was developed to perform free-radical-initiated peptide sequencing. Comparative collisional activation experiments (collision-induced dissociation and higher-energy collision-induced dissociation) with both cross-linkers were conducted in negative electrospray ionization mode with an Orbitrap Fusion mass spectrometer using five model peptides. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the loss of an intact peptide from the cross-linked products. As the BrPU linker combines a characteristic mass shift with an isotope signature, it presents a more favorable combination for automated assignment of cross-linked products compared with the TEMPO-Bz linker. [Figure not available: see fulltext.

  16. Templated Chemistry for Sequence-Specific Fluorogenic Detection of Duplex DNA

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    Li, Hao; Franzini, Raphael M.; Bruner, Christopher; Kool, Eric T.

    2015-01-01

    We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an α-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine, designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of ca. 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably, demonstrating high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and may allow detection of double stranded DNA, at least for homopurine-homopyrimidine target sites, under native, non-disturbing conditions. PMID:20859985

  17. cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

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    Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

    2011-02-04

    A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

  18. Synthesis, DNA Binding, and Anticancer Properties of Bis-Naphthalimide Derivatives with Lysine-Modified Polyamine Linkers

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    Yu Huang

    2018-01-01

    Full Text Available A series of bis-naphthalimide derivatives with different diamine linkers were designed and synthesized. All of the synthesized bis-naphthalimide derivatives were characterized by NMR and HRMS spectra. The binding ability between the compounds and CT DNA was evaluated by using UV–Vis titration experiments. The bis-naphthalimide compound with an ethylenediamine linker showed the largest binding constant with CT DNA. Hence, it was used as the model compound to study the DNA binding selectivity by UV–Vis titration aiming at different DNA duplexes. As a result, this compound showed binding preference to AT-rich duplexes. The DNA binding modes of the compounds were also measured by viscosity titration. The cytotoxicity of the compounds was evaluated by MTT assay. Compounds with 1,6-diaminohexane or 1,4-phenylenedimethanamine linkers showed higher cytotoxicity compared with other bis-naphthalimide derivatives.

  19. Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

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    Sarg, Bettina; Lopez, Rita; Lindner, Herbert; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-15

    establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative.

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    Scuotto, Maria; Rivieccio, Elisa; Varone, Alessia; Corda, Daniela; Bucci, Mariarosaria; Vellecco, Valentina; Cirino, Giuseppe; Virgilio, Antonella; Esposito, Veronica; Galeone, Aldo; Borbone, Nicola; Varra, Michela; Mayol, Luciano

    2015-09-18

    Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13. © Crown copyright 2015.

  1. Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

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    Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi

    2016-03-18

    Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remains unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.

  2. Crystallization of Galectin-8 Linker Reveals Intricate Relationship between the N-terminal Tail and the Linker

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    Yunlong Si

    2016-12-01

    Full Text Available Galectin-8 (Gal-8 plays a significant role in normal immunological function as well as in cancer. This lectin contains two carbohydrate recognition domains (CRD connected by a peptide linker. The N-terminal CRD determines ligand binding specificity, whereas the linker has been proposed to regulate overall Gal-8 function, including multimerization and biological activity. Here, we crystallized the Gal-8 N-terminal CRD with the peptide linker using a crystallization condition that contains Ni2+. The Ni2+ ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni2+ and Asp25 plays an indirect role in determining the structure of β-strand F0 and in influencing the linker conformation which could not be defined due to its dynamic nature. The linker was also shortened in situ and crystallized under a different condition, leading to a higher resolution structure refined to 1.08 Å. This crystal structure allowed definition of a short portion of the linker interacting with the Gal-8 N-terminal tail via ionic interactions and hydrogen bonds. Observation of two Gal-8 N-terminal CRD structures implies that the N-terminal tail and the linker may influence each other’s conformation. In addition, under specific crystallization conditions, glycerol could replace lactose and was observed at the carbohydrate binding site. However, glycerol did not show inhibition activity in hemagglutination assay.

  3. The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

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    Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

    2007-04-01

    The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

  4. Evaluation of ¹¹¹in-labelled exendin-4 derivatives containing different meprin β-specific cleavable linkers.

    Directory of Open Access Journals (Sweden)

    Andreas Jodal

    Full Text Available Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.The insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the 111In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound 111In-Ex4NOD40.In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.

  5. Desmosine-Inspired Cross-Linkers for Hyaluronan Hydrogels

    Science.gov (United States)

    Hagel, Valentin; Mateescu, Markus; Southan, Alexander; Wegner, Seraphine V.; Nuss, Isabell; Haraszti, Tamás; Kleinhans, Claudia; Schuh, Christian; Spatz, Joachim P.; Kluger, Petra J.; Bach, Monika; Tussetschläger, Stefan; Tovar, Günter E. M.; Laschat, Sabine; Boehm, Heike

    2013-06-01

    We designed bioinspired cross-linkers based on desmosine, the cross-linker in natural elastin, to prepare hydrogels with thiolated hyaluronic acid. These short, rigid cross-linkers are based on pyridinium salts (as in desmosine) and can connect two polymer backbones. Generally, the obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, have a large linear-elastic range, and show strain stiffening behavior typical for biopolymer networks. In addition, it is possible to introduce a positive charge to the core of the cross-linker without affecting the gelation efficiency, or consequently the network connectivity. However, the mechanical properties strongly depend on the charge of the cross-linker. The properties of the presented hydrogels can thus be tuned in a range important for engineering of soft tissues by controlling the cross-linking density and the charge of the cross-linker.

  6. Solid colloids with surface-mobile linkers

    International Nuclear Information System (INIS)

    Van der Meulen, Stef A J; Helms, Gesa; Dogterom, Marileen

    2015-01-01

    In this report we review the possibilities of using colloids with surface mobile linkers for the study of colloidal self-assembly processes. A promising route to create systems with mobile linkers is the use of lipid (bi-)layers. These lipid layers can be either used in the form of vesicles or as coatings for hard colloids and emulsion droplets. Inside the lipid bilayers molecules can be inserted via membrane anchors. Due to the fluidity of the lipid bilayer, the anchored molecules remain mobile. The use of different lipid mixtures even allows creating Janus-like particles that exhibit directional bonding if linkers are used which have a preference for a certain lipid phase. In nature mobile linkers can be found e.g. as receptors in cells. Therefore, towards the end of the review, we also briefly address the possibility of using colloids with surface mobile linkers as model systems to mimic cell–cell interactions and cell adhesion processes. (topical review)

  7. Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS.

    Science.gov (United States)

    Hage, Christoph; Ihling, Christian H; Götze, Michael; Schäfer, Mathias; Sinz, Andrea

    2017-01-01

    We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS 3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids. Graphical Abstract ᅟ.

  8. Structural Mechanisms of Nucleosome Recognition by Linker Histones.

    Science.gov (United States)

    Zhou, Bing-Rui; Jiang, Jiansheng; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T Sam; Bai, Yawen

    2015-08-20

    Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Hydroquinone–pyrrole dyads with varied linkers

    Directory of Open Access Journals (Sweden)

    Hao Huang

    2016-01-01

    Full Text Available A series of pyrroles functionalized in the 3-position with p-dimethoxybenzene via various linkers (CH2, CH2CH2, CH=CH, C≡C has been synthesized. Their electronic properties have been deduced from 1H NMR, 13C NMR, and UV–vis spectra to detect possible interactions between the two aromatic subunits. The extent of conjugation between the subunits is largely controlled by the nature of the linker, with the largest conjugation found with the trans-ethene linker and the weakest with the aliphatic linkers. DFT calculations revealed substantial changes in the HOMO–LUMO gap that correlated with the extent of conjugation found experimentally. The results of this work are expected to open up for use of the investigated compounds as components of redox-active materials in sustainable, organic electrical energy storage devices.

  10. Structure and Functions of Linker Histones.

    Science.gov (United States)

    Lyubitelev, A V; Nikitin, D V; Shaytan, A K; Studitsky, V M; Kirpichnikov, M P

    2016-03-01

    Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions - from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.

  11. Chimeric polyomavirus-derived virus-like particles: the immunogenicity of an inserted peptide applied without adjuvant to mice depends on its insertion site and its flanking linker sequence

    OpenAIRE

    Lawatscheck, R.; Aleksaite, E.; Schenk, J.A.; Micheel, B.; Jandrig, B.; Holland, G.; Sasnauskas, K.; Gedvilaite, A.; Ulrich, R.G.

    2007-01-01

    We inserted the sequence of the carcinoembryonic antigen-derived T cell epitope CAP-1-6D (CEA) into different positions of the hamster polyomavirus major capsid protein VP1. Independently from additional flanking linkers, yeast-expressed VP1 proteins harboring the CEA insertion between VP1 amino acid residues 80 and 89 (site 1) or 288 and 295 (site 4) or simultaneously at both positions assembled to chimeric virus-like particles (VLPs). BALB/c mice immunized with adjuvant-free VLPs developed ...

  12. Charged Triazole Cross-Linkers for Hyaluronan-Based Hybrid Hydrogels

    Directory of Open Access Journals (Sweden)

    Maike Martini

    2016-09-01

    Full Text Available Polyelectrolyte hydrogels play an important role in tissue engineering and can be produced from natural polymers, such as the glycosaminoglycan hyaluronan. In order to control charge density and mechanical properties of hyaluronan-based hydrogels, we developed cross-linkers with a neutral or positively charged triazole core with different lengths of spacer arms and two terminal maleimide groups. These cross-linkers react with thiolated hyaluronan in a fast, stoichiometric thio-Michael addition. Introducing a positive charge on the core of the cross-linker enabled us to compare hydrogels with the same interconnectivity, but a different charge density. Positively charged cross-linkers form stiffer hydrogels relatively independent of the size of the cross-linker, whereas neutral cross-linkers only form stable hydrogels at small spacer lengths. These novel cross-linkers provide a platform to tune the hydrogel network charge and thus the mechanical properties of the network. In addition, they might offer a wide range of applications especially in bioprinting for precise design of hydrogels.

  13. [Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

    Science.gov (United States)

    Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

    2015-12-01

    In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

  14. DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation.

    Science.gov (United States)

    Smaill, J B; Fan, J Y; Denny, W A

    1998-12-01

    A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with

  15. Initial conformation of kinesin's neck linker

    International Nuclear Information System (INIS)

    Geng Yi-Zhao; Yan Shi-Wei; Ji Qing; Liu Shu-Xia

    2014-01-01

    How ATP binding initiates the docking process of kinesin's neck linker is a key question in understanding kinesin mechanisms. By exploiting a molecular dynamics method, we investigate the initial conformation of kinesin's neck linker in its docking process. We find that, in the initial conformation, the neck linker has interactions with β0 and forms a ‘cover-neck bundle’ structure with β0. From this initial structure, the formation of extra turns and the docking of the cover-neck bundle structure can be achieved. The motor head provides a forward force on the initial cover-neck bundle structure through ATP-induced rotation. This force, together with the hydrophobic interaction of ILE327 with the hydrophobic pocket on the motor head, drives the formation of the extra turn and initiates the neck linker docking process. Based on these findings, a pathway from ATP binding-induced motor head rotation to neck linker docking is proposed. (interdisciplinary physics and related areas of science and technology)

  16. Nanohashtag structures based on carbon nanotubes and molecular linkers

    Science.gov (United States)

    Frye, Connor W.; Rybolt, Thomas R.

    2018-03-01

    Molecular mechanics was used to study the noncovalent interactions between single-walled carbon nanotubes and molecular linkers. Groups of nanotubes have the tendency to form tight, parallel bundles (||||). Molecular linkers were introduced into our models to stabilize nanostructures with carbon nanotubes held in perpendicular orientations. Molecular mechanics makes it possible to estimate the strength of noncovalent interactions holding these structures together and to calculate the overall binding energy of the structures. A set of linkers were designed and built around a 1,3,5,7-cyclooctatetraene tether with two corannulene containing pincers that extend in opposite directions from the central cyclooctatetraene portion. Each pincer consists of a pairs of "arms." These molecular linkers were modified so that the "hand" portions of each pair of "arms" could close together to grab and hold two carbon nanotubes in a perpendicular arrangement. To illustrate the possibility of more complicated and open perpendicular CNTs structures, our primary goal was to create a model of a nanohashtag (#) CNT conformation that is more stable than any parallel CNT arrangements with bound linker molecules forming clumps of CNTs and linkers in non-hashtag arrangements. This goal was achieved using a molecular linker (C280H96) that utilizes van der Waals interactions to two perpendicular oriented CNTs. Hydrogen bonding was then added between linker molecules to augment the stability of the hashtag structure. In the hashtag structure with hydrogen bonding, four (5,5) CNTs of length 4.46 nm (18 rings) and four linkers (C276H92N8O8) stabilized the hashtag so that the average binding energy per pincer was 118 kcal/mol.

  17. Lecithin-linker formulations for self-emulsifying delivery of nutraceuticals.

    Science.gov (United States)

    Chu, Jacquelene; Cheng, Yu-Ling; Rao, A Venketeshwer; Nouraei, Mehdi; Zarate-Muñoz, Silvia; Acosta, Edgar J

    2014-08-25

    Lecithin-linker microemulsions are formulations produced with soybean lecithin in combination with a highly lipophilic (lipophilic linker) and highly hydrophilic (hydrophilic linkers) surfactant-like additives. In this work, lecithin-linker systems were formulated to produce self-emulsifying delivery systems for β-carotene and β-sitosterol. The concentration of the lipophilic linker, sorbitan monooleate, was adjusted to minimize the formation of liquid crystals. The concentration of hydrophilic linkers, decaglyceryl caprylate/caprate and PEG-6-caprylic/capric glycerides, was gradually increased (scanned) until single phase clear microemulsions were obtained. For these scans, the oil (ethyl caprate) to water ratio was set to 1. The single phase, clear microemulsions were diluted with fed-state simulated intestinal fluid (FeSSIF) and produced stable emulsions, with drop sizes close to 200 nm. Using pseudo-ternary phase diagrams to evaluate the process of dilution of microemulsion preconcentrates (mixtures of oil, lecithin and linkers with little or no water) with FeSSIF, it was determined that self-emulsifying systems are obtained when the early stages of the dilution produce single phase microemulsions. If liquid crystals or multiple phase systems are obtained during those early stages, then the emulsification yields unstable emulsions with large drop sizes. An in vitro permeability study conducted using a Flow-Thru Dialyzer revealed that stable emulsions with drop sizes of 150-300 nm produce large and irreversible permeation of β-carotene to sheep intestine. On the other hand, unstable emulsions produced without the linker combination separated in the dialyzer chamber. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

    DEFF Research Database (Denmark)

    Rivas, Matilde De Las; Lira-Navarrete, Erandi; Daniel, Earnest James Paul

    2017-01-01

    The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation ...

  19. G-CSF receptor-binding cyclic peptides designed with artificial amino-acid linkers

    International Nuclear Information System (INIS)

    Shibata, Kenji; Maruyama-Takahashi, Kumiko; Yamasaki, Motoo; Hirayama, Noriaki

    2006-01-01

    Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with β-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay

  20. Photolabile linker for the synthesis of hydroxamic acids

    DEFF Research Database (Denmark)

    2013-01-01

    a hydroxylamine - functionalized photolabile linker, and the so produced hydroxylamine - functionalized photolabile solid support. The invention further provides a method for synthesizing a one-bead-one compound library of hydroxamic acid derivatives on a photolabile linker, as well as a method for screening...

  1. Mutations in B3GALT6, which Encodes a Glycosaminoglycan Linker Region Enzyme, Cause a Spectrum of Skeletal and Connective Tissue Disorders

    OpenAIRE

    Nakajima, Masahiro; Mizumoto, Shuji; Miyake, Noriko; Kogawa, Ryo; Iida, Aritoshi; Ito, Hironori; Kitoh, Hiroshi; Hirayama, Aya; Mitsubuchi, Hiroshi; Miyazaki, Osamu; Kosaki, Rika; Horikawa, Reiko; Lai, Angeline; Mendoza-Londono, Roberto; Dupuis, Lucie

    2013-01-01

    Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a sever...

  2. Improving the performance of DomainDiscovery of protein domain boundary assignment using inter-domain linker index

    Directory of Open Access Journals (Sweden)

    Zomaya Albert Y

    2006-12-01

    Full Text Available Abstract Background Knowledge of protein domain boundaries is critical for the characterisation and understanding of protein function. The ability to identify domains without the knowledge of the structure – by using sequence information only – is an essential step in many types of protein analyses. In this present study, we demonstrate that the performance of DomainDiscovery is improved significantly by including the inter-domain linker index value for domain identification from sequence-based information. Improved DomainDiscovery uses a Support Vector Machine (SVM approach and a unique training dataset built on the principle of consensus among experts in defining domains in protein structure. The SVM was trained using a PSSM (Position Specific Scoring Matrix, secondary structure, solvent accessibility information and inter-domain linker index to detect possible domain boundaries for a target sequence. Results Improved DomainDiscovery is compared with other methods by benchmarking against a structurally non-redundant dataset and also CASP5 targets. Improved DomainDiscovery achieves 70% accuracy for domain boundary identification in multi-domains proteins. Conclusion Improved DomainDiscovery compares favourably to the performance of other methods and excels in the identification of domain boundaries for multi-domain proteins as a result of introducing support vector machine with benchmark_2 dataset.

  3. Linker-mediated assembly of gold nanoparticles into multimeric motifs

    Energy Technology Data Exchange (ETDEWEB)

    Sikora, Mateusz; Cieplak, Marek [Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46, 02-668 Warsaw (Poland); Szymczak, Piotr [Institute of Theoretical Physics, Faculty of Physics, University of Warsaw, ulica Hoza 69, 00-681 Warsaw (Poland); Thompson, Damien, E-mail: mc@ifpan.edu.pl [Tyndall National Institute, Lee Maltings, University College Cork, Cork (Ireland)

    2011-11-04

    We present a theoretical description of linker-mediated self-assembly of gold nanoparticles (Au-NP). Using mesoscale simulations with a coarse-grained model for the Au NPs and dirhenium-based linker molecules, we investigate the conditions under which large clusters can grow and construct a phase diagram that identifies favorable growth conditions in terms of floating and bound linker concentrations. The findings can be considered as generic, as we expect other NP-linker systems to behave in a qualitatively similar way. In particular, we also discuss the case of antibody-functionalised Au NPs connected by the C-reactive proteins (CRPs). We extract some general rules for NP linking that may aid the production of size- and shape-specific NP clusters for technology applications.

  4. Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

    Science.gov (United States)

    Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

    2016-08-02

    The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Yeast linker histone Hho1p is required for efficient RNA polymerase I processivity and transcriptional silencing at the ribosomal DNA

    OpenAIRE

    Levy, Anat; Eyal, Miri; Hershkovits, Gitit; Salmon-Divon, Mali; Klutstein, Michael; Katcoff, Don Jay

    2008-01-01

    Nucleosome core particles in eukaryotes are linked by a stretch of DNA that is usually associated with a linker histone. Here, we show in yeast, that the presence of yeast linker histone Hho1p represses expression of a pol II transcribed gene (MET15) embedded in the rDNA. In vivo deletions of Hho1p sequences showed that the second globular domain is sufficient for that repression, whereas the presence of the N terminus is required for its derepression. In contrast, a run-on assay confirmed by...

  6. Construction of hierarchically porous metal-organic frameworks through linker labilization

    Science.gov (United States)

    Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; Li, Jialuo; Huang, Lan; Feng, Liang; Wang, Xuan; Bosch, Mathieu; Alsalme, Ali; Cagin, Tahir; Zhou, Hong-Cai

    2017-05-01

    A major goal of metal-organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. Herein, we present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragments by acid treatment. We demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.

  7. Improved sensitivity of a graphene FET biosensor using porphyrin linkers

    Science.gov (United States)

    Kawata, Takuya; Ono, Takao; Kanai, Yasushi; Ohno, Yasuhide; Maehashi, Kenzo; Inoue, Koichi; Matsumoto, Kazuhiko

    2018-06-01

    Graphene FET (G-FET) biosensors have considerable potential due to the superior characteristics of graphene. Realizing this potential requires judicious choice of the linker molecule connecting the target-specific receptor molecule to the graphene surface, yet there are few reports comparing linker molecules for G-FET biosensors. In this study, tetrakis(4-carboxyphenyl)porphyrin (TCPP) was used as a linker for surface modification of a G-FET and the properties of the device were compared to those of a G-FET device modified with the conventional linker 1-pyrenebutanoic acid succinimidyl ester (PBASE). TCPP modification resulted in a higher density of receptor immunoglobulin E (IgE) aptamer molecules on the G-FET. The detection limit of the target IgE was enhanced from 13 nM for the PBASE-modified G-FET to 2.2 nM for the TCPP-modified G-FET, suggesting that the TCPP linker is a powerful candidate for G-FET modification.

  8. A Traceless Aryl-Triazene Linker for DNA-Directed Chemistry

    DEFF Research Database (Denmark)

    Hejesen, Christian; Pedersen, Lars Kolster; Gothelf, Kurt Vesterager

    2013-01-01

    DNA-directed synthesis of encoded combinatorial libraries of small organic compounds most often involves transfer of organic building blocks from one DNA strand to another. This requires cleavable linkers to enable cleavage of the link to the original DNA strand from which the building block...... is transferred. Relatively few cleavable linkers are available for DNA-directed synthesis and most often they leave an amino group at the organic molecule. Here we have extended the application of 10 aryltriazenes as traceless linkers for DNA-directed synthesis. After reaction of one building block...

  9. Sequence-specific DNA alkylation by tandem Py-Im polyamide conjugates.

    Science.gov (United States)

    Taylor, Rhys Dylan; Kawamoto, Yusuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2014-09-01

    Tandem N-methylpyrrole-N-methylimidazole (Py-Im) polyamides with good sequence-specific DNA-alkylating activities have been designed and synthesized. Three alkylating tandem Py-Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high-resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1, which contained a β-alanine linker, displayed the most-selective sequence-specific alkylation towards the target 10 bp DNA sequence. The tandem Py-Im polyamide conjugates displayed greater sequence-specific DNA alkylation than conventional hairpin Py-Im polyamide conjugates (4 and 5). For further research, the design of tandem Py-Im polyamide conjugates could play an important role in targeting specific gene sequences. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Creating Hierarchical Pores by Controlled Linker Thermolysis in Multivariate Metal-Organic Frameworks.

    Science.gov (United States)

    Feng, Liang; Yuan, Shuai; Zhang, Liang-Liang; Tan, Kui; Li, Jia-Luo; Kirchon, Angelo; Liu, Ling-Mei; Zhang, Peng; Han, Yu; Chabal, Yves J; Zhou, Hong-Cai

    2018-02-14

    Sufficient pore size, appropriate stability, and hierarchical porosity are three prerequisites for open frameworks designed for drug delivery, enzyme immobilization, and catalysis involving large molecules. Herein, we report a powerful and general strategy, linker thermolysis, to construct ultrastable hierarchically porous metal-organic frameworks (HP-MOFs) with tunable pore size distribution. Linker instability, usually an undesirable trait of MOFs, was exploited to create mesopores by generating crystal defects throughout a microporous MOF crystal via thermolysis. The crystallinity and stability of HP-MOFs remain after thermolabile linkers are selectively removed from multivariate metal-organic frameworks (MTV-MOFs) through a decarboxylation process. A domain-based linker spatial distribution was found to be critical for creating hierarchical pores inside MTV-MOFs. Furthermore, linker thermolysis promotes the formation of ultrasmall metal oxide nanoparticles immobilized in an open framework that exhibits high catalytic activity for Lewis acid-catalyzed reactions. Most importantly, this work provides fresh insights into the connection between linker apportionment and vacancy distribution, which may shed light on probing the disordered linker apportionment in multivariate systems, a long-standing challenge in the study of MTV-MOFs.

  11. Backbone amide linker strategy

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen

    2013-01-01

    In the backbone amide linker (BAL) strategy, the peptide is anchored not at the C-terminus but through a backbone amide, which leaves the C-terminal available for various modifications. This is thus a very general strategy for the introduction of C-terminal modifications. The BAL strategy...

  12. Rational design of molecularly imprinted polymer: the choice of cross-linker.

    Science.gov (United States)

    Muhammad, Turghun; Nur, Zohre; Piletska, Elena V; Yimit, Osmanjan; Piletsky, Sergey A

    2012-06-07

    The paper describes a rational approach for the selection of cross-linkers during the development of molecularly imprinted polymers (MIPs). As a model system for this research MIPs specific for the drug zidovudine (AZT) were designed and tested. Three cross-linkers trimethylolpropane trimethacrylate (TRIM), ethylene glycol dimethacrylate (EGDMA) and divinylbenzene (DVB) were studied. The analogue of zidovudine (AZT) ester (AZT-ES) was used as a dummy template. The imprinting factors for all of the polymers in the static adsorption experiments were calculated. The data on the AZT adsorption by control polymers (CP), which were prepared with different cross-linkers without a functional monomer, was also analyzed. DVB was found to be more inert towards zidovudine than EGDMA and TRIM, which was confirmed by both molecular modelling and adsorption experiments. It was demonstrated that DVB-based polymers had a higher imprinting factor (I = 1.85) compared with other tested cross-linked polymers. It was suggested that the selection of the cross-linker should be based on the strength of the interaction with the template: the cross-linker which displays lower binding of the template should be preferential because it generates MIPs with lower non-specific binding and a higher imprinting factor, and therefore specificity. Which cross-linker to use for the preparation of any particular MIP can be determined by analysis of the interactions between the cross-linker and template. This could be done either virtually using computational modelling or by template adsorption using a small library of polymers prepared using different cross-linkers.

  13. The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

    Science.gov (United States)

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

    2014-01-01

    T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

  14. Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

    Science.gov (United States)

    Belotserkovskii, Boris P; Soo Shin, Jane Hae; Hanawalt, Philip C

    2017-06-20

    Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practical implications for the design of expression vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. A Helix-Stabilizing Linker Improves Subcutaneous Bioavailability of a Helical Peptide Independent of Linker Lipophilicity

    Science.gov (United States)

    Zhang, Liang; Navaratna, Tejas; Thurber, Greg M.

    2016-01-01

    Stabilized peptides address several limitations to peptide-based imaging agents and therapeutics such as poor stability and low affinity due to conformational flexibility. There is also active research in developing these compounds for intracellular drug targeting, and significant efforts have been invested to determine the effects of helix stabilization on intracellular delivery. However, much less is known about the impact on other pharmacokinetic parameters such as plasma clearance and bioavailability. We investigated the effect of different fluorescent helix-stabilizing linkers with varying lipophilicity on subcutaneous (SC) bioavailability using the glucagon-like peptide-1 (GLP-1) receptor ligand exendin as a model system. The stabilized peptides showed significantly higher protease resistance and increased bioavailability independent of linker hydrophilicity, and all subcutaneously delivered conjugates were able to successfully target the islets of Langerhans with high specificity. The lipophilic peptide variants had slower absorption and plasma clearance than their respective hydrophilic conjugates, and the absolute bioavailability was also lower likely due to the longer residence times in the skin. The ease and efficiency of double-click helix stabilization chemistries is a useful tool for increasing the bioavailability of peptide therapeutics, many of which suffer from rapid in vivo protease degradation. Helix stabilization using linkers of varying lipophilicity can further control SC absorption and clearance rates to customize plasma pharmacokinetics. PMID:27327034

  16. Creating Hierarchical Pores by Controlled Linker Thermolysis in Multivariate Metal-Organic Frameworks

    KAUST Repository

    Feng, Liang

    2018-01-18

    Sufficient pore size, appropriate stability and hierarchical porosity are three prerequisites for open frameworks designed for drug delivery, enzyme immobilization and catalysis involving large molecules. Herein, we report a powerful and general strate-gy, linker thermolysis, to construct ultra-stable hierarchically porous metal−organic frameworks (HP-MOFs) with tunable pore size distribution. Linker instability, usually an undesirable trait of MOFs, was exploited to create mesopores by generating crystal defects throughout a microporous MOF crystal via thermolysis. The crystallinity and stability of HP-MOFs remain after thermolabile linkers are selectively removed from multivariate metal-organic frameworks (MTV-MOFs) through a decarboxyla-tion process. A domain-based linker spatial distribution was found to be critical for creating hierarchical pores inside MTV-MOFs. Furthermore, linker thermolysis promotes the formation of ultra-small metal oxide (MO) nanoparticles immobilized in an open framework that exhibits high catalytic activity for Lewis acid catalyzed reactions. Most importantly, this work pro-vides fresh insights into the connection between linker apportionment and vacancy distribution, which may shed light on prob-ing the disordered linker apportionment in multivariate systems, a long-standing challenge in the study of MTV-MOFs.

  17. Open and closed: the roles of linker histones in plants and animals.

    Science.gov (United States)

    Over, Ryan S; Michaels, Scott D

    2014-03-01

    Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications (PTMs) to core histones, but linker histone function is less well understood. In this review, we describe the current understanding of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and development, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.

  18. The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

    Science.gov (United States)

    Sean, Polen; Nguyen, Joseph H C; Semler, Bert L

    2008-09-01

    Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

  19. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    Directory of Open Access Journals (Sweden)

    Bharti Arvind K

    2008-12-01

    Full Text Available Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR and methylation spanning linker libraries (MSLL. These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig, while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%. These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of

  20. Differential solvation of intrinsically disordered linkers drives the formation of spatially organized droplets in ternary systems of linear multivalent proteins

    Science.gov (United States)

    Harmon, Tyler S.; Holehouse, Alex S.; Pappu, Rohit V.

    2018-04-01

    Intracellular biomolecular condensates are membraneless organelles that encompass large numbers of multivalent protein and nucleic acid molecules. The bodies assemble via a combination of liquid–liquid phase separation and gelation. A majority of condensates included multiple components and show multilayered organization as opposed to being well-mixed unitary liquids. Here, we put forward a simple thermodynamic framework to describe the emergence of spatially organized droplets in multicomponent systems comprising of linear multivalent polymers also known as associative polymers. These polymers, which mimic proteins and/or RNA have the architecture of domains or motifs known as stickers that are interspersed by flexible spacers known as linkers. Using a minimalist numerical model for a four-component system, we have identified features of linear multivalent molecules that are necessary and sufficient for generating spatially organized droplets. We show that differences in sequence-specific effective solvation volumes of disordered linkers between interaction domains enable the formation of spatially organized droplets. Molecules with linkers that are preferentially solvated are driven to the interface with the bulk solvent, whereas molecules that have linkers with negligible effective solvation volumes form cores in the core–shell architectures that emerge in the minimalist four-component systems. Our modeling has relevance for understanding the physical determinants of spatially organized membraneless organelles.

  1. Surface expression and subunit specific control of steady protein levels by the Kv7.2 helix A-B linker.

    Directory of Open Access Journals (Sweden)

    Paloma Aivar

    Full Text Available Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A - helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.

  2. A streptavidin linker layer that functions after drying.

    Science.gov (United States)

    Xia, Nan; Shumaker-Parry, Jennifer S; Zareie, M Hadi; Campbell, Charles T; Castner, David G

    2004-04-27

    The ability of streptavidin (SA) to simultaneously bind four biotins is often used in linker layers, where a biotinylated molecule is linked to a biotin-functionalized surface via SA. For biosensor and array applications, it is desirable that the SA linker layer be stable to drying and rehydration. In this study it was observed that a significant decrease in binding capacity of a SA layer occurred when that layer was dried. For this study a SA linker layer was constructed by binding SA to a biotin-containing alkylthiolate monolayer (BAT/OEG) self-assembled onto gold. Its stability after drying was investigated using surface plasmon resonance (SPR). Approximately a quarter of the SA layer was removed from the BAT/OEG surface upon drying and rehydration, suggesting disruption of SA-biotin binding when dry. This resulted in the dried SA layer losing approximately 40% of its biotinylated ferritin (BF) binding capacity. Coating the layer with trehalose before drying was found to inhibit the loss of SA from the BAT/OEG surface. SPR showed that the trehalose-protected SA linker layer retained approximately 91% of its original BF binding capacity after drying and rehydration. Atomic force microscopy, which was used to image individual surface-bound SA and BF molecules, qualitatively confirmed these observations.

  3. Investigation of the Linker Swing Motion in the Zeolitic Imidazolate Framework ZIF-90

    KAUST Repository

    Zheng, Bin

    2018-03-13

    The linker swing motion in the zeolitic imidazolate framework ZIF-90 is investigated by density functional theory (DFT) calculation, molecular dynamics (MD) and grand-canonical Monte Carlo (GCMC) simulations. The relation between the terminal aldehyde group rotation and the linker swing motion is revealed. The extremely high activation energy of the linker swing motion in ZIF-90 can be attributed to the asymmetric geometry and electron distribution of aldehyde groups. The change in the gate structure resulting from the linker rotation is used to understand the guest adsorption in ZIF-90. This study shows that it is possible to tune the linker swing motion and then the properties of ZIF-90 by manipulating the terminal group rotation. The results highlight the importance of considering the internal freedom effects to correctly describe the linker swing motion and the flexibility of metal-organic frameworks (MOFs).

  4. Sequencing and analysis of the gene-rich space of cowpea

    Directory of Open Access Journals (Sweden)

    Cheung Foo

    2008-02-01

    Full Text Available Abstract Background Cowpea, Vigna unguiculata (L. Walp., is one of the most important food and forage legumes in the semi-arid tropics because of its drought tolerance and ability to grow on poor quality soils. Approximately 80% of cowpea production takes place in the dry savannahs of tropical West and Central Africa, mostly by poor subsistence farmers. Despite its economic and social importance in the developing world, cowpea remains to a large extent an underexploited crop. Among the major goals of cowpea breeding and improvement programs is the stacking of desirable agronomic traits, such as disease and pest resistance and response to abiotic stresses. Implementation of marker-assisted selection and breeding programs is severely limited by a paucity of trait-linked markers and a general lack of information on gene structure and organization. With a nuclear genome size estimated at ~620 Mb, the cowpea genome is an ideal target for reduced representation sequencing. Results We report here the sequencing and analysis of the gene-rich, hypomethylated portion of the cowpea genome selectively cloned by methylation filtration (MF technology. Over 250,000 gene-space sequence reads (GSRs with an average length of 610 bp were generated, yielding ~160 Mb of sequence information. The GSRs were assembled, annotated by BLAST homology searches of four public protein annotation databases and four plant proteomes (A. thaliana, M. truncatula, O. sativa, and P. trichocarpa, and analyzed using various domain and gene modeling tools. A total of 41,260 GSR assemblies and singletons were annotated, of which 19,786 have unique GenBank accession numbers. Within the GSR dataset, 29% of the sequences were annotated using the Arabidopsis Gene Ontology (GO with the largest categories of assigned function being catalytic activity and metabolic processes, groups that include the majority of cellular enzymes and components of amino acid, carbohydrate and lipid metabolism. A

  5. Dipolar cross-linkers for PDMS networks with enhanced dielectric permittivity and low dielectric loss

    DEFF Research Database (Denmark)

    Bahrt, Frederikke; Daugaard, Anders Egede; Hvilsted, Søren

    2013-01-01

    -(4-((4-nitrophenyl)diazenyl)phenoxy)-prop-1-yn-1-ylium, with a synthesized silicone compatible azide-functional cross-linker by click chemistry. The thermal, mechanical and electromechanical properties were investigated for PDMS films with 0 to 3.6 wt% of dipole-cross-linker. The relative dielectric permittivity......Dipole grafted cross-linkers were utilized to prepare polydimethylsiloxane (PDMS) elastomers with various chain lengths and with various concentrations of functional cross-linker. The grafted cross-linkers were prepared by reaction of two alkyne-functional dipoles, 1-ethynyl-4-nitrobenzene and 3...

  6. Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

    1996-02-15

    Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U).

  7. Monte Carlo analysis of neck linker extension in kinesin molecular motors.

    Directory of Open Access Journals (Sweden)

    Matthew L Kutys

    2010-11-01

    Full Text Available Kinesin stepping is thought to involve both concerted conformational changes and diffusive movement, but the relative roles played by these two processes are not clear. The neck linker docking model is widely accepted in the field, but the remainder of the step--diffusion of the tethered head to the next binding site--is often assumed to occur rapidly with little mechanical resistance. Here, we investigate the effect of tethering by the neck linker on the diffusive movement of the kinesin head, and focus on the predicted behavior of motors with naturally or artificially extended neck linker domains. The kinesin chemomechanical cycle was modeled using a discrete-state Markov chain to describe chemical transitions. Brownian dynamics were used to model the tethered diffusion of the free head, incorporating resistive forces from the neck linker and a position-dependent microtubule binding rate. The Brownian dynamics and chemomechanical cycle were coupled to model processive runs consisting of many 8 nm steps. Three mechanical models of the neck linker were investigated: Constant Stiffness (a simple spring, Increasing Stiffness (analogous to a Worm-Like Chain, and Reflecting (negligible stiffness up to a limiting contour length. Motor velocities and run lengths from simulated paths were compared to experimental results from Kinesin-1 and a mutant containing an extended neck linker domain. When tethered by an increasingly stiff spring, the head is predicted to spend an unrealistically short amount of time within the binding zone, and extending the neck is predicted to increase both the velocity and processivity, contrary to experiments. These results suggest that the Worm-Like Chain is not an adequate model for the flexible neck linker domain. The model can be reconciled with experimental data if the neck linker is either much more compliant or much stiffer than generally assumed, or if weak kinesin-microtubule interactions stabilize the diffusing

  8. Synthetic incorporation of Nile Blue into DNA using 2′-deoxyriboside substitutes: Representative comparison of (R- and (S-aminopropanediol as an acyclic linker

    Directory of Open Access Journals (Sweden)

    Daniel Lachmann

    2010-02-01

    Full Text Available The Nile Blue chromophore was incorporated into oligonucleotides using “click” chemistry for the postsynthetic modification of oligonucleotides. These were synthesized using DNA building block 3 bearing an alkyne group and reacted with the azide 4. (R-3-amino-1,2-propanediol was applied as the linker between the phosphodiester bridges. Two sets of DNA duplexes were prepared. One set carried the chromophore in an A-T environment, the second set in a G-C environment. Both were characterized by optical spectroscopy. Sequence-dependent fluorescence quenching was applied as a sensitive tool to compare the stacking interactions with respect to the chirality of the acyclic linker attachment. The results were compared to recent results from duplexes that carried the Nile Blue label in a sequentially and structurally identical context, except for the opposite chirality of the linker ((S-3-amino-1,2-propandiol. Only minor, negligible differences were observed. Melting temperatures, UV–vis absorption spectra together with fluorescence quenching data indicate that Nile Blue stacks perfectly between the adjacent base pairs regardless of whether it has been attached via an S- or R-configured linker. This result was supported by geometrically optimized DNA models.

  9. Linkers, resins, and general procedures for solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen

    2013-01-01

    and linkers for solid-phase synthesis is a key parameter for successful peptide synthesis. This chapter provides an overview of the most common and useful resins and linkers for the synthesis of peptides with C-terminal amides, carboxylic acids, and more. The chapter finishes with robust protocols for general...

  10. Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation

    Science.gov (United States)

    Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira

    2018-01-01

    Background Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. Methods We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. Results In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. Conclusions These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers. PMID:29629343

  11. LRRC45 Is a Centrosome Linker Component Required for Centrosome Cohesion

    Directory of Open Access Journals (Sweden)

    Runsheng He

    2013-09-01

    Full Text Available During interphase, centrosomes are connected by a proteinaceous linker between the proximal ends of the centrioles, which is important for the centrosomes to function as a single microtubule-organizing center. However, the composition and regulation of centrosomal linker remain largely unknown. Here, we show that LRRC45 is a centrosome linker that localizes at the proximal ends of the centrioles and forms fiber-like structures between them. Depletion of LRRC45 results in centrosome splitting during interphase. Moreover, LRRC45 interacts with both C-Nap1 and rootletin and is phosphorylated by Nek2A at S661 during mitosis. After phosphorylation, both LRRC45 centrosomal localization and fiber-like structures are significantly reduced, which subsequently leads to centrosome separation. Thus, LRRC45 is a critical component of the proteinaceous linker between two centrioles and is required for centrosome cohesion.

  12. Water-soluble heterobifunctional fluorescent linkers

    Czech Academy of Sciences Publication Activity Database

    Bartoň, Jan; Cígler, Petr

    2017-01-01

    Roč. 15, č. 1 (2017), s. 4 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : fluorescent probes * heterobifunctional linkers Subject RIV: CA - Inorganic Chemistry

  13. Library of biphenyl privileged substructures using a safety-catch linker approach

    DEFF Research Database (Denmark)

    Severinsen, Rune; Bourne, Gregory T; Tran, Tran T

    2008-01-01

    A biphenyl privileged structure library containing three attachment points were synthesized using a catechol-based safety-catch linker strategy. The method requires the attachment of a bromo-acid to the linker, followed by a Pd-catalyzed Suzuki cross-coupling reaction. Further derivatization...

  14. Chirality- and sequence-selective successive self-sorting via specific homo- and complementary-duplex formations

    Science.gov (United States)

    Makiguchi, Wataru; Tanabe, Junki; Yamada, Hidekazu; Iida, Hiroki; Taura, Daisuke; Ousaka, Naoki; Yashima, Eiji

    2015-01-01

    Self-recognition and self-discrimination within complex mixtures are of fundamental importance in biological systems, which entirely rely on the preprogrammed monomer sequences and homochirality of biological macromolecules. Here we report artificial chirality- and sequence-selective successive self-sorting of chiral dimeric strands bearing carboxylic acid or amidine groups joined by chiral amide linkers with different sequences through homo- and complementary-duplex formations. A mixture of carboxylic acid dimers linked by racemic-1,2-cyclohexane bis-amides with different amide sequences (NHCO or CONH) self-associate to form homoduplexes in a completely sequence-selective way, the structures of which are different from each other depending on the linker amide sequences. The further addition of an enantiopure amide-linked amidine dimer to a mixture of the racemic carboxylic acid dimers resulted in the formation of a single optically pure complementary duplex with a 100% diastereoselectivity and complete sequence specificity stabilized by the amidinium–carboxylate salt bridges, leading to the perfect chirality- and sequence-selective duplex formation. PMID:26051291

  15. Creating Hierarchical Pores by Controlled Linker Thermolysis in Multivariate Metal-Organic Frameworks

    KAUST Repository

    Feng, Liang; Yuan, Shuai; Zhang, Liang-Liang; Tan, Kui; Li, Jia-Luo; Kirchon, Angelo; Liu, Ling-Mei; Zhang, Peng; Han, Yu; Chabal, Yves J.; Zhou, Hong-Cai

    2018-01-01

    strate-gy, linker thermolysis, to construct ultra-stable hierarchically porous metal−organic frameworks (HP-MOFs) with tunable pore size distribution. Linker instability, usually an undesirable trait of MOFs, was exploited to create mesopores

  16. Tunable CO 2 Adsorbents by Mixed-Linker Synthesis and Postsynthetic Modification of Zeolitic Imidazolate Frameworks

    KAUST Repository

    Thompson, Joshua A.; Brunelli, Nicholas A.; Lively, Ryan P.; Johnson, J. R.; Jones, Christopher W.; Nair, Sankar

    2013-01-01

    The incorporation of accessible amine functionality in zeolitic imidazolate frameworks (ZIFs) is used to improve the adsorption selectivity for CO 2/CH4 gas separation applications. Two synthetic approaches are described in this work to introduce functionality into the ZIF: (i) mixed-linker ZIF synthesis with 2-aminobenzimidazole as a substitution linker and (ii) postsynthetic modification of a mixed-linker ZIF with ethylenediamine. Using 2-aminobenzimidazole, a linker with a primary amine functional group, substitution of the ZIF-8 linker during synthesis allows for control over the adsorption properties while maintaining the ZIF-8 structure with up to nearly 50% substitution in the mixed-linker ZIF framework, producing a material with tunable pore size and amine functionality. Alternatively, postsynthetic modification of a mixed-linker ZIF containing an aldehyde functional group produces a ZIF material with a primary amine without detrimental loss of micropore volume by controlling the amount of functional group sites for modification. Both approaches using mixed-linker ZIFs yield new materials that show improvement in adsorption selectivity for the CO 2/CH4 gas pair over ZIF-8 and commercially available adsorbents as well as an increase in the heat of adsorption for CO2 without significant changes to the crystal structure. These results indicate that tuning the surface properties of ZIFs by either mixed-linker synthesis and/or postsynthetic modification may generate new materials with improved gas separation properties, thereby providing a new method for tailoring metal-organic frameworks. © 2013 American Chemical Society.

  17. Tunable CO 2 Adsorbents by Mixed-Linker Synthesis and Postsynthetic Modification of Zeolitic Imidazolate Frameworks

    KAUST Repository

    Thompson, Joshua A.

    2013-04-25

    The incorporation of accessible amine functionality in zeolitic imidazolate frameworks (ZIFs) is used to improve the adsorption selectivity for CO 2/CH4 gas separation applications. Two synthetic approaches are described in this work to introduce functionality into the ZIF: (i) mixed-linker ZIF synthesis with 2-aminobenzimidazole as a substitution linker and (ii) postsynthetic modification of a mixed-linker ZIF with ethylenediamine. Using 2-aminobenzimidazole, a linker with a primary amine functional group, substitution of the ZIF-8 linker during synthesis allows for control over the adsorption properties while maintaining the ZIF-8 structure with up to nearly 50% substitution in the mixed-linker ZIF framework, producing a material with tunable pore size and amine functionality. Alternatively, postsynthetic modification of a mixed-linker ZIF containing an aldehyde functional group produces a ZIF material with a primary amine without detrimental loss of micropore volume by controlling the amount of functional group sites for modification. Both approaches using mixed-linker ZIFs yield new materials that show improvement in adsorption selectivity for the CO 2/CH4 gas pair over ZIF-8 and commercially available adsorbents as well as an increase in the heat of adsorption for CO2 without significant changes to the crystal structure. These results indicate that tuning the surface properties of ZIFs by either mixed-linker synthesis and/or postsynthetic modification may generate new materials with improved gas separation properties, thereby providing a new method for tailoring metal-organic frameworks. © 2013 American Chemical Society.

  18. Open and Closed: The Roles of Linker Histones in Plants and Animals

    OpenAIRE

    Over, Ryan S.; Michaels, Scott D.

    2014-01-01

    Linker histones play key roles alongside core histones in the regulation and maintenance of chromatin. Here, we illustrate our current understanding of the contributions of linker histones to the cell cycle, development, and chromatin structure in plants and animals.

  19. Novel silicone compatible cross-linkers for controlled functionalization of PDMS networks

    DEFF Research Database (Denmark)

    Madsen, Frederikke Bahrt; Daugaard, Anders Egede; Hvilsted, Søren

    2013-01-01

    . In order to improve the dielectric properties of PDMS a novel system is developed where push-pull dipoles are grafted to a new silicone compatible cross-linker. The grafted cross-linkers are prepared by reaction of two different push-pull dipole alkynes as well as a fluorescent alkyne with the new azide...

  20. Preferential 5-Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein.

    Science.gov (United States)

    Kizaki, Seiichiro; Zou, Tingting; Li, Yue; Han, Yong-Woon; Suzuki, Yuki; Harada, Yoshie; Sugiyama, Hiroshi

    2016-11-07

    Tet (ten-eleven translocation) family proteins oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG-methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Directing the breathing behavior of pillared-layered metal-organic frameworks via a systematic library of functionalized linkers bearing flexible substituents.

    Science.gov (United States)

    Henke, Sebastian; Schneemann, Andreas; Wütscher, Annika; Fischer, Roland A

    2012-06-06

    Flexible metal-organic frameworks (MOFs), also referred to as soft porous crystals (SPCs), show reversible structural transitions dependent on the nature and quantity of adsorbed guest molecules. In recent studies it has been reported that covalent functionalization of the organic linker can influence or even integrate framework flexibility ("breathing") in MOFs. However, rational fine-tuning of such responsive properties is very desirable but challenging as well. Here we present a powerful approach for the targeted manipulation of responsiveness and framework flexibility of an important family of pillared-layered MOFs based on the parent structure [Zn(2)(bdc)(2)(dabco)](n) (bdc = 1,4-benzenedicarboxylate; dabco = 1,4-diazabicyclo[2.2.2]octane). A library of functionalized bdc-type linkers (fu-bdc), which bear additional dangling side groups at different positions of the benzene core (alkoxy groups of varying chain length with diverse functionalities and polarity), was generated. Synthesis of the materials [Zn(2)(fu-bdc)(2)(dabco)](n) yields the respective collection of highly responsive MOFs. The parent MOF is only weakly flexible; however, the substituted frameworks of [Zn(2)(fu-bdc)(2)(dabco)](n) contract drastically upon guest removal and expand again upon adsorption of DMF (N,N-dimethylformamide), EtOH, or CO(2), etc., while N(2) is hardly adsorbed and does not open the narrow-pored form. These "breathing" dynamics are attributed to the dangling side chains that act as immobilized "guests", which interact with mobile guest molecules as well as with themselves and with the framework backbone. The structural details of the guest-free, contracted form and the gas sorption behavior (phase transition pressure, hysteresis loop) are highly dependent on the nature of the substituent at the linker and can therefore be adjusted using our approach. Combining our library of functionalized linkers with the concept of mixed-component MOFs (solid solutions) offers very rich

  2. The measles virus phosphoprotein interacts with the linker domain of STAT1

    International Nuclear Information System (INIS)

    Devaux, Patricia; Priniski, Lauren; Cattaneo, Roberto

    2013-01-01

    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway

  3. The measles virus phosphoprotein interacts with the linker domain of STAT1

    Energy Technology Data Exchange (ETDEWEB)

    Devaux, Patricia, E-mail: devaux.patricia@mayo.edu; Priniski, Lauren; Cattaneo, Roberto

    2013-09-15

    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.

  4. Development of Bioorthogonally Degradable Linkers and Polymers Using alpha-Azidoethers

    Science.gov (United States)

    Rajagopalan, Chandrasekhar Ramasubramanian

    Degradable polymers have gained a lot of attention in recent years for applications in biotechnology and medicine. External control over polymer degradation can be obtained by incorporating functional groups that cleave in the presence of triggers that would normally be absent in biological environments, i.e. are bioorthogonal. This thesis explores the use of chemically cleavable alpha-azidoethers as a new method to obtain external control over the degradation behavior of polymers. My first goal is to illustrate the potential of alpha-azidoethers toward developing cleavable linkers. We have studied the relationship between alpha-azidoether structure and hydrolytic stability, to prepare linkers that withstand background hydrolytic cleavage until they are exposed to the cleaving trigger. The cleavage kinetics of the alpha-azidoether functional group was quantified. In addition to the conventionally used tris(2-carboxyethyl)phosphine (TCEP), dihydrolipoic acid (DHLA), a previously unexplored, biocompatible reducing agent, was also evaluated as a cleaving trigger. Based on these results, we have proposed design rules for utilizing alpha-azidoethers as cleavable linkers in applications that require bioorthogonal control over linker cleavage. Secondly, the alpha-azidoether cleavable linker chemistry was implemented into the development of polymeric materials. Two different types of polymers were developed. Polyamides incorporating alpha-azidoethers along the backbone were synthesized, and their physical properties and chemically triggered degradation behavior were characterized. The degradation timescale of these polymers can be tuned simply by manipulating the concentration of the externally applied chemical trigger. The alpha-azidoether functional group was then utilized to develop a unique triggered-release polymeric adhesive for potential applications in dental adhesive formulations. A methacrylamide-phosphonate adhesive monomer incorporating an alpha

  5. A Small Number of Residues Can Determine if Linker Histones Are Bound On or Off Dyad in the Chromatosome.

    Science.gov (United States)

    Zhou, Bing-Rui; Feng, Hanqiao; Ghirlando, Rodolfo; Li, Shipeng; Schwieters, Charles D; Bai, Yawen

    2016-10-09

    Linker histones bind to the nucleosome and regulate the structure and function of chromatin. We have previously shown that the globular domains of chicken H5 and Drosophila H1 linker histones bind to the nucleosome with on- or off-dyad modes, respectively. To explore the determinant for the distinct binding modes, we investigated the binding of a mutant globular domain of H5 to the nucleosome. This mutant, termed GH5_pMut, includes substitutions of five globular domain residues of H5 with the corresponding residues in the globular domain of Drosophila H1. The residues at these five positions play important roles in nucleosome binding by either H5 or Drosophila H1. NMR and spin-labeling experiments showed that GH5_pMut bound to the nucleosome off the dyad. We further found that the nucleosome array condensed by either the GH5_pMut or the globular domain of Drosophila H1 displayed a similar sedimentation coefficient, whereas the same nucleosome array condensed by the wild-type globular domain of H5 showed a much larger sedimentation coefficient. Moreover, NMR and spin-labeling results from the study of the nucleosome in complex with the full-length human linker histone H1.0, whose globular domain shares high sequence conservation with the corresponding globular domain of H5, are consistent with an on-dyad binding mode. Taken together, our results suggest that a small number of residues in the globular domain of a linker histone can control its binding location on the nucleosome and higher-order chromatin structure. Copyright © 2016. Published by Elsevier Ltd.

  6. Composite materials with metal oxide attached to lead chalcogenide nanocrystal quantum dots with linkers

    Science.gov (United States)

    Fuke, Nobuhiro; Koposov, Alexey Y; Sykora, Milan; Hoch, Laura

    2014-12-16

    Composite materials useful for devices such as photoelectrochemical solar cells include a substrate, a metal oxide film on the substrate, nanocrystalline quantum dots (NQDs) of lead sulfide, lead selenide, and lead telluride, and linkers that attach the NQDs to the metal oxide film. Suitable linkers preserve the 1s absorption peak of the NQDs. A suitable linker has a general structure A-B-C where A is a chemical group adapted for binding to a MO.sub.x and C is a chemical group adapted for binding to a NQD and B is a divalent, rigid, or semi-rigid organic spacer moiety. Other linkers that preserve the 1s absorption peak may also be used.

  7. Use of designed sequences in protein structure recognition.

    Science.gov (United States)

    Kumar, Gayatri; Mudgal, Richa; Srinivasan, Narayanaswamy; Sandhya, Sankaran

    2018-05-09

    Knowledge of the protein structure is a pre-requisite for improved understanding of molecular function. The gap in the sequence-structure space has increased in the post-genomic era. Grouping related protein sequences into families can aid in narrowing the gap. In the Pfam database, structure description is provided for part or full-length proteins of 7726 families. For the remaining 52% of the families, information on 3-D structure is not yet available. We use the computationally designed sequences that are intermediately related to two protein domain families, which are already known to share the same fold. These strategically designed sequences enable detection of distant relationships and here, we have employed them for the purpose of structure recognition of protein families of yet unknown structure. We first measured the success rate of our approach using a dataset of protein families of known fold and achieved a success rate of 88%. Next, for 1392 families of yet unknown structure, we made structural assignments for part/full length of the proteins. Fold association for 423 domains of unknown function (DUFs) are provided as a step towards functional annotation. The results indicate that knowledge-based filling of gaps in protein sequence space is a lucrative approach for structure recognition. Such sequences assist in traversal through protein sequence space and effectively function as 'linkers', where natural linkers between distant proteins are unavailable. This article was reviewed by Oliviero Carugo, Christine Orengo and Srikrishna Subramanian.

  8. Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

    Science.gov (United States)

    Ott, Wolfgang; Jobst, Markus A; Bauer, Magnus S; Durner, Ellis; Milles, Lukas F; Nash, Michael A; Gaub, Hermann E

    2017-06-27

    Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

  9. A High-Throughput Small Molecule Screen for C. elegans Linker Cell Death Inhibitors.

    Directory of Open Access Journals (Sweden)

    Andrew R Schwendeman

    Full Text Available Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery.

  10. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    Science.gov (United States)

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Linker-dependent Junction Formation Probability in Single-Molecule Junctions

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Pil Sun; Kim, Taekyeong [HankukUniversity of Foreign Studies, Yongin (Korea, Republic of)

    2015-01-15

    We compare the junction formation probabilities of single-molecule junctions with different linker molecules by using a scanning tunneling microscope-based break-junction technique. We found that the junction formation probability varies as SH > SMe > NH2 for the benzene backbone molecule with different types of anchoring groups, through quantitative statistical analysis. These results are attributed to different bonding forces according to the linker groups formed with Au atoms in the electrodes, which is consistent with previous works. Our work allows a better understanding of the contact chemistry in the metal.molecule junction for future molecular electronic devices.

  12. The leucine-rich repeat structure.

    Science.gov (United States)

    Bella, J; Hindle, K L; McEwan, P A; Lovell, S C

    2008-08-01

    The leucine-rich repeat is a widespread structural motif of 20-30 amino acids with a characteristic repetitive sequence pattern rich in leucines. Leucine-rich repeat domains are built from tandems of two or more repeats and form curved solenoid structures that are particularly suitable for protein-protein interactions. Thousands of protein sequences containing leucine-rich repeats have been identified by automatic annotation methods. Three-dimensional structures of leucine-rich repeat domains determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. As the essential structural principles become well established, the leucine-rich repeat architecture is emerging as an attractive framework for structural prediction and protein engineering. This review presents an update of the current understanding of leucine-rich repeat structure at the primary, secondary, tertiary and quaternary levels and discusses specific examples from recently determined three-dimensional structures.

  13. Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

    Science.gov (United States)

    Alvarado, John J; Betts, Laurie; Moroco, Jamie A; Smithgall, Thomas E; Yeh, Joanne I

    2010-11-12

    Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a "conformational switch" that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that "fine-tune" their sensitivities to activation by SH3-based ligands.

  14. Mixed-linker zeolitic imidazolate framework mixed-matrix membranes for aggressive CO2 separation from natural gas

    KAUST Repository

    Thompson, Joshua A.

    2014-07-01

    Zeolitic imidazolate framework (ZIF) materials are a promising subclass of metal-organic frameworks (MOF) for gas separations. However, due to the deleterious effects of gate-opening phenomena associated with organic linker rotation near the limiting pore apertures of ZIFs, there have been few demonstrations of improved gas separation properties over pure polymer membranes when utilizing ZIF materials in composite membranes for CO2-based gas separations. Here, we report a study of composite ZIF/polymer membranes, containing mixed-linker ZIF materials with ZIF-8 crystal topologies but composed of different organic linker compositions. Characterization of the mixed-linker ZIFs shows that the mixed linker approach offers control over the porosity and pore size distribution of the materials, as determined from nitrogen physisorption and Horváth-Kawazoe analysis. Single gas permeation measurements on mixed-matrix membranes reveal that inclusion of mixed-linker ZIFs yields membranes with better ideal CO2/CH4 selectivity than membranes containing ZIF-8. This improvement is shown to likely occur from enhancement in the diffusion selectivity of the membranes associated with controlling the pore size distribution of the ZIF filler. Mixed-gas permeation experiments show that membranes with mixed-linker ZIFs display an effective plasticization resistance that is not typical of the pure polymeric matrix. Overall, we demonstrate that mixed-linker ZIFs can improve the gas separation properties in composite membranes and may be applicable to aggressive CO2 concentrations in natural gas feeds. © 2013 Elsevier Inc. All rights reserved.

  15. High-Flux Zeolitic Imidazolate Framework Membranes for Propylene/Propane Separation by Postsynthetic Linker Exchange.

    Science.gov (United States)

    Lee, Moon Joo; Kwon, Hyuk Taek; Jeong, Hae-Kwon

    2018-01-02

    While zeolitic imidazolate framework, ZIF-8, membranes show impressive propylene/propane separation, their throughput needs to be greatly improved for practical applications. A method is described that drastically reduces the effective thickness of ZIF-8 membranes, thereby substantially improving their propylene permeance (that is, flux). The new strategy is based on a controlled single-crystal to single-crystal linker exchange of 2-methylimidazole in ZIF-8 membrane grains with 2-imidazolecarboxaldehyde (ZIF-90 linker), thereby enlarging the effective aperture size of ZIF-8. The linker-exchanged ZIF-8 membranes showed a drastic increase in propylene permeance by about four times, with a negligible loss in propylene/propane separation factor when compared to as-prepared membranes. The linker-exchange effect depends on the membrane synthesis method. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

    Science.gov (United States)

    Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

    2010-10-01

    Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

  17. Role of H1 linker histones in mammalian development and stem cell differentiation.

    Science.gov (United States)

    Pan, Chenyi; Fan, Yuhong

    2016-03-01

    H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Effect of linkers on the αvβ3 integrin targeting efficiency of cyclic RGD-conjugates

    Science.gov (United States)

    Karmakar, Partha; Grabowska, Dorota; Sudlow, Gail; Ziabrev, Kostiantyn; Sanyal, Nibedita; Achilefu, Samuel

    2018-02-01

    Cyclic arginine-glycine-aspartic acid (cRGD) peptides are well known to target ανβ3 integrin expressed on cancer cells and neovasculature. Conjugation of these peptides with dyes, drugs, antibodies and other biomolecules through covalent linkers provides a facile way to deliver these products to tumor cells for targeted cancer therapy and diagnosis. Click chemistry and acid-amine couplings are widely used conjugation strategies. However, the effects of different linkers and the distance between the cRGD and the conjugates on the binding of cRGD ligand with ανβ3 has been underexplored. In this present study, we prepared cRGD-conjugates using different linkers and determined how they altered the tumor targeting efficiency in vitro and in vivo. The results demonstrate that different linkers significantly altered the pharmacokinetics of the cRGD conjugates and the tumor uptake kinetics. Unlike large antibodies, this preliminary finding shows that linkers used to attach drugs and fluorescent molecular probes to small peptides play a major role in the accuracy of tumor targeting and treatment outcomes. As a result, considerable attention should be paid to the nature of linkers used in the design of molecular probes and targeted therapeutics.

  19. The centrosomal linker and microtubules provide dual levels of spatial coordination of centrosomes.

    Directory of Open Access Journals (Sweden)

    Marko Panic

    2015-05-01

    Full Text Available The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm. Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.

  20. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    International Nuclear Information System (INIS)

    Zhang, Yuan; Palla, Mirkó; Liao, Jung-Chi; Sun, Andrew

    2013-01-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116. (paper)

  1. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    Science.gov (United States)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  2. Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing*S⃞

    Science.gov (United States)

    Thiriet, Christophe; Hayes, Jeffrey J.

    2009-01-01

    Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing. PMID:19015270

  3. G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

    Science.gov (United States)

    Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

    2016-01-01

    Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. PMID:27471032

  4. Specific distribution of the Saccharomyces cerevisiae linker histone homolog HHO1p in the chromatin

    OpenAIRE

    Freidkin, Ilya; Katcoff, Don J.

    2001-01-01

    In virtually all eukaryotic organisms, linker DNA between nucleosomes is associated with a histone termed linker histone or histone H1. In Saccharomyces cerevisiae, HHO1 encodes a putative linker histone with very significant homology to histone H1. The encoded protein is expressed in the nucleus, but has not been shown to affect global chromatin structure, nor has its deletion shown any detectable phenotype. In vitro chromatin assembly experiments with recombinant HHO1p have shown that it is...

  5. Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks

    Science.gov (United States)

    Belotserkovskii, Boris P.; Neil, Alexander J.; Saleh, Syed Shayon; Shin, Jane Hae Soo; Mirkin, Sergei M.; Hanawalt, Philip C.

    2013-01-01

    The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816–12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation. PMID:23275544

  6. Chemical and enzymatic stability of amino acid prodrugs containing methoxy, ethoxy and propylene glycol linkers.

    Science.gov (United States)

    Gupta, Deepak; Gupta, Sheeba Varghese; Lee, Kyung-Dall; Amidon, Gordon L

    2009-01-01

    We evaluated the chemical and enzymatic stabilities of prodrugs containing methoxy, ethoxy and propylene glycol linkers in order to find a suitable linker for prodrugs of carboxylic acids with amino acids. l-Valine and l-phenylalanine prodrugs of model compounds (benzoic acid and phenyl acetic acid) containing methoxy, ethoxy and propylene glycol linkers were synthesized. The hydrolysis rate profile of each compound was studied at physiologically relevant pHs (1.2, 4, 6 and 7.4). Enzymatic hydrolysis of propylene glycol containing compounds was studied using Caco-2 homogenate as well as purified enzyme valacyclovirase. It was observed that the stability of the prodrugs increases with the linker length (propyl > ethyl > methyl). The model prodrugs were stable at acidic pH as compared to basic pH. It was observed that the prodrug with the aliphatic amino acid promoiety was more stable compared to its aromatic counterpart. The comparison between benzyl and the phenyl model compounds revealed that the amino acid side chain is significant in determining the stability of the prodrug whereas the benzyl or phenyl carboxylic acid had little or no effect on the stability. The enzymatic activation studies of propylene glycol linker prodrug in the presence of valacyclovirase and cell homogenate showed faster generation of the parent drug at pH 7.4. The half-life of prodrugs at pH 7.4 was more than 12 h, whereas in the presence of cell homogenate the half-lives were less than 1 h. Hydrolysis by Caco-2 homogenate generated the parent compound in two steps, where the prodrug was first converted to the intermediate, propylene glycol benzoate, which was then converted to the parent compound (benzoic acid). Enzymatic hydrolysis of propylene glycol containing prodrugs by valacyclovirase showed hydrolysis of the amino acid ester part to generate the propylene glycol ester of model compound (propylene glycol benzoate) as the major product. The amino acid prodrugs containing methoxy

  7. Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

    Science.gov (United States)

    Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

    2017-01-01

    Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

  8. The flexibility of modified-linker MIL-53 materials.

    Science.gov (United States)

    Munn, Alexis S; Pillai, Renjith S; Biswas, Shyam; Stock, Norbert; Maurin, Guillaume; Walton, Richard I

    2016-03-14

    The flexibility of eight aluminium hydroxo terephthalates [Al(OH)(BDC-X)]·n(guest) (BDC = 1,4-benzene-dicarboxylate; X = -H, -CH3, -Cl, -Br, -NH2, -NO2, -(OH)2, -CO2H) crystallising in the MIL-53-type structure was investigated upon thermal dehydration of as-made samples, superhydration and methanol adsorption/desorption using in situ powder X-ray diffraction (PXRD). Profile fitting was used to determine lattice parameters as a function of time and/or temperature to describe their structural evolution. It has thus been shown that while methanol vapour adsorption induces an opening of all the modified frameworks, except the -NH2 material, superhydration only leads to open structures for Al-MIL-53-NO2, -Br and -(OH)2. All the MIL-53 solids, except Al-MIL-53-(OH)2 are present in the open structures upon thermal dehydration. In addition to the exploration of the breathing behavior of this MIL-53 series, the issue of disorder in the distribution of the functional groups between the organic linkers was explored. As a typical illustration, density functional theory calculations were carried out on different structures of Al-MIL-53-Cl, in which the distribution of -Cl within two adjacent BDC linkers is varied. The results show that the most energetically stable configuration leads to the best agreement with the experimental PXRD pattern. This observation supports that the distribution of the selected linker substituent in the functionalised solid is governed by energetics and that there is a preference for an ordering of this arrangement.

  9. Rapid One-Pot Microwave Synthesis of Mixed-Linker Hybrid Zeolitic-Imidazolate Framework Membranes for Tunable Gas Separations.

    Science.gov (United States)

    Hillman, Febrian; Brito, Jordan; Jeong, Hae-Kwon

    2018-02-14

    The relatively slow and complex fabrication processes of polycrystalline metal-organic framework (MOF) membranes often times restrict their way to commercialization, despite their potential for molecular separation applications. Herein, we report a rapid one-pot microwave synthesis of mixed-linker hybrid zeolitic-imidazolate framework (ZIF) membranes consisting of 2-methylimidazolate (ZIF-8 linker) and benzimidazolate (ZIF-7 linker) linkers, termed ZIF-7-8 membranes. The fast-volumetric microwave heating in conjunction with a unique counter diffusion of metal and linker solutions enabled unprecedented rapid synthesis of well-intergrown ZIF-7-8 membranes in ∼90 s, the fastest MOF membrane preparation up to date. Furthermore, we were able to tune the molecular sieving properties of the ZIF-7-8 membranes by varying the benzimidazole-to-2-methylimidazole (bIm-to-mIm) linker ratio in the hybrid frameworks. The tuning of their molecular sieving properties led to the systematic change in the permeance and selectivity of various small gases. The unprecedented rapid synthesis of well-intergrown ZIF-7-8 membranes with tunable molecular sieving properties is an important step forward for the commercial gas separation applications of ZIF membranes.

  10. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.

    Science.gov (United States)

    Yan, Qingrong; Barros, Tiago; Visperas, Patrick R; Deindl, Sebastian; Kadlecek, Theresa A; Weiss, Arthur; Kuriyan, John

    2013-06-01

    Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.

  11. Synthesis of Selective Butyrylcholinesterase Inhibitors Coupled between α-Lipoic Acid and Polyphenols by Using 2-(Piperazin-1-yl)ethanol Linker

    International Nuclear Information System (INIS)

    Yeun, Go Heun; Lee, Seung Hwan; LIm, Yong Bae; Lee, Hye Sook; Lee, Bong Ho; Park, Jeong Ho; Won, Mooho

    2013-01-01

    In the previous paper (Bull. Korean Chem. Soc., 2011, 32, 2997), the hybrid molecules between α-lipoic acid (ALA) and polyphenols (PPs) connected with neutral 2-(2-aminoethoxy)ethanol linker (linker-1) showed new biological activity such as butyrylcholinesterase (BuChE) inhibition. In order to increase the binding affinity of the hybrid compounds to cholinesterase (ChE), the neutral 2-(2-aminoethoxy)ethanol (linker 1) was switched to the cationic 2-(piperazin-1-yl)ethanol linker (linker 2). The IC 50 values of the linker-2 hybrid molecules for BuChE inhibition were lower than those of linker-1 hybrid molecules (except 9-2) and they also had the same great selectivity for BuChE over AChE (> 800 fold) as linker-1 hybrid molecules. ALA-acetyl caffeic acid (10-2, ALA-AcCA) was shown as an effective inhibitor of BuChE (IC 50 = 0.44 ± 0.24 μM). A kinetic study using 7-2 showed that it is the same mixed type inhibition as 7-1. Its inhibition constant (Ki) to BuChE is 4.3 ± 0.09 μM

  12. Synthesis of Selective Butyrylcholinesterase Inhibitors Coupled between α-Lipoic Acid and Polyphenols by Using 2-(Piperazin-1-yl)ethanol Linker

    Energy Technology Data Exchange (ETDEWEB)

    Yeun, Go Heun; Lee, Seung Hwan; LIm, Yong Bae; Lee, Hye Sook; Lee, Bong Ho; Park, Jeong Ho [Hanbat National Univ., Daejeon (Korea, Republic of); Won, Mooho [Kangwon National Univ., Chuncheon (Korea, Republic of)

    2013-04-15

    In the previous paper (Bull. Korean Chem. Soc., 2011, 32, 2997), the hybrid molecules between α-lipoic acid (ALA) and polyphenols (PPs) connected with neutral 2-(2-aminoethoxy)ethanol linker (linker-1) showed new biological activity such as butyrylcholinesterase (BuChE) inhibition. In order to increase the binding affinity of the hybrid compounds to cholinesterase (ChE), the neutral 2-(2-aminoethoxy)ethanol (linker 1) was switched to the cationic 2-(piperazin-1-yl)ethanol linker (linker 2). The IC{sub 50} values of the linker-2 hybrid molecules for BuChE inhibition were lower than those of linker-1 hybrid molecules (except 9-2) and they also had the same great selectivity for BuChE over AChE (> 800 fold) as linker-1 hybrid molecules. ALA-acetyl caffeic acid (10-2, ALA-AcCA) was shown as an effective inhibitor of BuChE (IC{sub 50} = 0.44 ± 0.24 μM). A kinetic study using 7-2 showed that it is the same mixed type inhibition as 7-1. Its inhibition constant (Ki) to BuChE is 4.3 ± 0.09 μM.

  13. Regulation of Cellular Dynamics and Chromosomal Binding Site Preference of Linker Histones H1.0 and H1.X.

    Science.gov (United States)

    Okuwaki, Mitsuru; Abe, Mayumi; Hisaoka, Miharu; Nagata, Kyosuke

    2016-11-01

    Linker histones play important roles in the genomic organization of mammalian cells. Of the linker histone variants, H1.X shows the most dynamic behavior in the nucleus. Recent research has suggested that the linker histone variants H1.X and H1.0 have different chromosomal binding site preferences. However, it remains unclear how the dynamics and binding site preferences of linker histones are determined. Here, we biochemically demonstrated that the DNA/nucleosome and histone chaperone binding activities of H1.X are significantly lower than those of other linker histones. This explains why H1.X moves more rapidly than other linker histones in vivo Domain swapping between H1.0 and H1.X suggests that the globular domain (GD) and C-terminal domain (CTD) of H1.X independently contribute to the dynamic behavior of H1.X. Our results also suggest that the N-terminal domain (NTD), GD, and CTD cooperatively determine the preferential binding sites, and the contribution of each domain for this determination is different depending on the target genes. We also found that linker histones accumulate in the nucleoli when the nucleosome binding activities of the GDs are weak. Our results contribute to understanding the molecular mechanisms of dynamic behaviors, binding site selection, and localization of linker histones. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. P-Link: A method for generating multicomponent cytochrome P450 fusions with variable linker length

    DEFF Research Database (Denmark)

    Belsare, Ketaki D.; Ruff, Anna Joelle; Martinez, Ronny

    2014-01-01

    Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P...

  15. Direct selection of expressed sequences on a YAC clone revealed proline-rich-like genes and BARE-1 sequences physically linked to the complex ¤Mla¤ powdery mildew resistance locus of barley (¤Hordeum vulgare¤ L.)

    DEFF Research Database (Denmark)

    Schwarz, G.; Michalek, W.; Jahoor, A.

    2002-01-01

    homology to the copia-like retroelement BA REI of barley, putatively involved in evolution of disease resistance loci. The high degree of clones representing barley rRNA sequences or false positives is a major disadvantage of direct selection of cDNAs in barley. (C) 2002 Elsevier Science Ireland Ltd. All...... gene. Of 22 selected cDNA clones, six were re-located on the YAC by southern analysis. Two of these clones are predicted to encode members of the hydroxyproline-rich glycoprotein and proline-rich protein gene families which have been implicated in plant defense response. Four sequences showed high...

  16. Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

    Science.gov (United States)

    Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang

    2016-01-21

    Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Tethering metal ions to photocatalyst particulate surfaces by bifunctional molecular linkers for efficient hydrogen evolution

    KAUST Repository

    Yu, Weili

    2014-08-19

    A simple and versatile method for the preparation of photocatalyst particulates modified with effective cocatalysts is presented; the method involves the sequential soaking of photocatalyst particulates in solutions containing bifunctional organic linkers and metal ions. The modification of the particulate surfaces is a universal and reproducible method because the molecular linkers utilize strong covalent bonds, which in turn result in modified monolayer with a small but controlled quantity of metals. The photocatalysis results indicated that the CdS with likely photochemically reduced Pd and Ni, which were initially immobilized via ethanedithiol (EDT) as a linker, were highly efficient for photocatalytic hydrogen evolution from Na2S-Na2SO3-containing aqueous solutions. The method developed in this study opens a new synthesis route for the preparation of effective photocatalysts with various combinations of bifunctional linkers, metals, and photocatalyst particulate materials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Tethering metal ions to photocatalyst particulate surfaces by bifunctional molecular linkers for efficient hydrogen evolution

    KAUST Repository

    Yu, Weili; Isimjan, Tayirjan T.; Del Gobbo, Silvano; Anjum, Dalaver Hussain; Abdel-Azeim, Safwat; Cavallo, Luigi; Garcia Esparza, Angel T.; Domen, Kazunari; Xu, Wei; Takanabe, Kazuhiro

    2014-01-01

    A simple and versatile method for the preparation of photocatalyst particulates modified with effective cocatalysts is presented; the method involves the sequential soaking of photocatalyst particulates in solutions containing bifunctional organic linkers and metal ions. The modification of the particulate surfaces is a universal and reproducible method because the molecular linkers utilize strong covalent bonds, which in turn result in modified monolayer with a small but controlled quantity of metals. The photocatalysis results indicated that the CdS with likely photochemically reduced Pd and Ni, which were initially immobilized via ethanedithiol (EDT) as a linker, were highly efficient for photocatalytic hydrogen evolution from Na2S-Na2SO3-containing aqueous solutions. The method developed in this study opens a new synthesis route for the preparation of effective photocatalysts with various combinations of bifunctional linkers, metals, and photocatalyst particulate materials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Robust Optical Richness Estimation with Reduced Scatter

    Energy Technology Data Exchange (ETDEWEB)

    Rykoff, E.S.; /LBL, Berkeley; Koester, B.P.; /Chicago U. /Chicago U., KICP; Rozo, E.; /Chicago U. /Chicago U., KICP; Annis, J.; /Fermilab; Evrard, A.E.; /Michigan U. /Michigan U., MCTP; Hansen, S.M.; /Lick Observ.; Hao, J.; /Fermilab; Johnston, D.E.; /Fermilab; McKay, T.A.; /Michigan U. /Michigan U., MCTP; Wechsler, R.H.; /KIPAC, Menlo Park /SLAC

    2012-06-07

    Reducing the scatter between cluster mass and optical richness is a key goal for cluster cosmology from photometric catalogs. We consider various modifications to the red-sequence matched filter richness estimator of Rozo et al. (2009b), and evaluate their impact on the scatter in X-ray luminosity at fixed richness. Most significantly, we find that deeper luminosity cuts can reduce the recovered scatter, finding that {sigma}{sub ln L{sub X}|{lambda}} = 0.63 {+-} 0.02 for clusters with M{sub 500c} {approx}> 1.6 x 10{sup 14} h{sub 70}{sup -1} M{sub {circle_dot}}. The corresponding scatter in mass at fixed richness is {sigma}{sub ln M|{lambda}} {approx} 0.2-0.3 depending on the richness, comparable to that for total X-ray luminosity. We find that including blue galaxies in the richness estimate increases the scatter, as does weighting galaxies by their optical luminosity. We further demonstrate that our richness estimator is very robust. Specifically, the filter employed when estimating richness can be calibrated directly from the data, without requiring a-priori calibrations of the red-sequence. We also demonstrate that the recovered richness is robust to up to 50% uncertainties in the galaxy background, as well as to the choice of photometric filter employed, so long as the filters span the 4000 {angstrom} break of red-sequence galaxies. Consequently, our richness estimator can be used to compare richness estimates of different clusters, even if they do not share the same photometric data. Appendix A includes 'easy-bake' instructions for implementing our optimal richness estimator, and we are releasing an implementation of the code that works with SDSS data, as well as an augmented maxBCG catalog with the {lambda} richness measured for each cluster.

  20. The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair

    Science.gov (United States)

    Plys, Aaron J.; Rogacheva, Maria V.; Greene, Eric C.; Alani, Eric

    2012-01-01

    DNA mismatch repair (MMR) models have proposed that MSH proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH proteins (primarily Mlh1-Pms1 in baker’s yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20 – 30 nanometers) unstructured arms that connect two terminal globular domains. These arms can vary between 100 to 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker’s yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR. PMID:22659005

  1. Synthesis and evaluation of sequence-specific DNA alkylating agents: effect of alkylation subunits.

    Science.gov (United States)

    Shimizu, Tatsuhiko; Sasaki, Shunta; Minoshima, Masafumi; Shinohara, Ken-ichi; Bando, Toshikazu; Sugiyama, Hiroshi

    2006-01-01

    We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

  2. Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation.

    Science.gov (United States)

    Findeisen, Felix; Minor, Daniel L

    2009-03-01

    Two processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

  3. pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

    Science.gov (United States)

    Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

    2009-11-23

    Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

  4. In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

    Science.gov (United States)

    Tanaka, Mizuki; Sakai, Yoshifumi; Yamada, Osamu; Shintani, Takahiro; Gomi, Katsuya

    2011-01-01

    To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them. PMID:21586533

  5. A photolabile linker for the solid-phase synthesis of 4-substituted NH-1,2,3-triazoles

    DEFF Research Database (Denmark)

    Qvortrup, Katrine; Nielsen, Thomas Eiland

    2011-01-01

    A novel photolabile linker for solid-phase synthesis is presented. The linker displays an azido handle for copper-catalyzed azide–alkyne cycloaddition reactions with a variety of alkynes, remains intact under typical solid-phase reaction conditions, and enables a mild photolytic release of 4...

  6. A Photolabile Linker for the Solid-Phase Synthesis of Peptide Hydrazides and Heterocycles

    DEFF Research Database (Denmark)

    Qvortrup, Katrine; Komnatnyy, Vitaly V.; Nielsen, Thomas Eiland

    2014-01-01

    A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard amino adds, including those with side......-chain protected residues at the C-terminal of the resulting peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most commonly used coupling reagents and protecting groups for solid-phase peptide synthesis....

  7. Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells.

    Science.gov (United States)

    Gao, Yunfeng; Foo, Yong Hwee; Winardhi, Ricksen S; Tang, Qingnan; Yan, Jie; Kenney, Linda J

    2017-11-21

    Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

  8. How to remain nonfolded and pliable: the linkers in modular α-amylases as a case study.

    Science.gov (United States)

    Feller, Georges; Dehareng, Dominique; Lage, Jean-Luc Da

    2011-07-01

    The primary structure of linkers in a new class of modular α-amylases constitutes a paradigm of the structural basis that allows a polypeptide to remain nonfolded, extended and pliable. Unfolding is mediated through a depletion of hydrophobic residues and an enrichment of hydrophilic residues, amongst which Ser and Thr are over-represented. An extended and flexible conformation is promoted by the sequential arrangement of Pro and Gly, which are the most abundant residues in these linkers. This is complemented by charge repulsion, charge clustering and disulfide-bridged loops. Molecular dynamics simulations suggest the existence of conformational transitions resulting from a transient and localized hydrophobic collapse, arising from the peculiar composition of the linkers. Accordingly, these linkers should not be regarded as fully disordered, but rather as possessing various discrete structural patterns allowing them to fulfill their biological function as a free energy reservoir for concerted motions between structured domains. © 2011 The Authors Journal compilation © 2011 FEBS.

  9. DNA sequence-specific dimeric bisbenzimidazoles DBP(n) and DBPA(n) as inhibitors of H-NS silencing in bacterial cells.

    Science.gov (United States)

    Melkina, Olga E; Koval, Vasilii S; Ivanov, Alexander A; Zhuze, Alexei L; Zavilgelsky, Gennadii B

    2018-03-01

    DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns + . The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5μM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters. Copyright © 2017 Elsevier GmbH. All rights reserved.

  10. Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6.

    Science.gov (United States)

    Kim, Han Ie; Jung, Jinwon; Lee, Eun-Saem; Kim, Yong-Chul; Lee, Weontae; Lee, Seung-Taek

    2007-11-03

    PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3-Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3-Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

  11. Fivefold increase of hydrogen uptake in MOF74 through linker decorations

    Science.gov (United States)

    Arter, C. A.; Zuluaga, S.; Harrison, D.; Welchman, E.; Thonhauser, T.

    2016-10-01

    We present ab initio results for linker decorations in Mg-MOF74, i.e., attaching various metals M =Li, Na, K, Sc, Cr, Mn, Fe, Ni, Cu, Zn, Rb, Pd, Ag, and Pt near the ring of the linker, creating new strong adsorption sites and thus maximizing small-molecule uptake. We find that in most cases these decorations influence the overall form and structure of Mg-MOF74 only marginally. After the initial screening, we chose metals that bind favorably to the linker and further investigated adsorption of H2,CO2, and H2O for M =Li , Na, K, and Sc. For the case of H2 we show that up to 24 additional guest molecules can be adsorbed in the metal-organic framework (MOF) unit cell, with binding energies comparable to the original open-metal sites at the six corners of the channel. This leads to a fivefold increase of the molecule uptake in Mg-MOF74, with tremendous impact on many applications in general and hydrogen storage in particular, where the gravimetric hydrogen density increases from 1.63 to 7.28 mass % and the volumetric density increases from 15.10 to 75.50 g H2L-1 .

  12. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    OpenAIRE

    Chang, Keejong; Qian, Jin; Jiang, MeiSheng; Liu, Yi-Hsin; Wu, Ming-Che; Chen, Chi-Dar; Lai, Chao-Kuen; Lo, Hsin-Lung; Hsiao, Chin-Ton; Brown, Lucy; Bolen, James; Huang, Hsiao-I; Ho, Pei-Yu; Shih, Ping Yao; Yao, Chen-Wen

    2002-01-01

    Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal ...

  13. ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1*

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-01-01

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer. PMID:20007714

  14. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-02-12

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  15. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James

    2004-01-01

    Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

  16. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R.; Foulongne-Oriol, Marie; Lombard, Vincent; Nagy, Laszlo G.; Ohm, Robin A.; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L.; Bailey, Andrew M.; Billette, Christophe; Coutinho, Pedro M.; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hilden, Kristiina; Kues, Ursula; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Murat, Claude; Riley, Robert W.; Salamov, Asaf A.; Schmutz, Jeremy; Subramanian, Venkataramanan; Wosten, Han A. B.; Xu, Jianping; Eastwood, Daniel C.; Foster, Gary D.; Sonnenberg, Anton S. M.; Cullen, Dan; de Vries, Ronald P.; Lundell, Taina; Hibbett, David S.; Henrissat, Bernard; Burton, Kerry S.; Kerrigan, Richard W.; Challen, Michael P.; Grigoriev, Igor V.; Martin, Francis

    2012-04-27

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the button mushroom forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

  17. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

    Science.gov (United States)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R; Foulongne-Oriol, Marie; Lombard, Vincent; Nagy, Laszlo G; Ohm, Robin A; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L; Bailey, Andrew M; Billette, Christophe; Coutinho, Pedro M; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hildén, Kristiina; Kües, Ursula; Labutti, Kurt M; Lapidus, Alla; Lindquist, Erika A; Lucas, Susan M; Murat, Claude; Riley, Robert W; Salamov, Asaf A; Schmutz, Jeremy; Subramanian, Venkataramanan; Wösten, Han A B; Xu, Jianping; Eastwood, Daniel C; Foster, Gary D; Sonnenberg, Anton S M; Cullen, Dan; de Vries, Ronald P; Lundell, Taina; Hibbett, David S; Henrissat, Bernard; Burton, Kerry S; Kerrigan, Richard W; Challen, Michael P; Grigoriev, Igor V; Martin, Francis

    2012-10-23

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

  18. Novel mixing method for cross linker introduction into droplet emulsions

    CSIR Research Space (South Africa)

    Land, KJ

    2013-10-01

    Full Text Available the introduction of cross linker after droplet formation, together with the utilisation of topological microfluidic channel structures, allowing for the novel manufacture of particles. Flow over these structures has been simulated in order to choose the most...

  19. One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

    Science.gov (United States)

    Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

    2011-09-12

    A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

  20. Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity.

    Science.gov (United States)

    Sindbert, Simon; Kalinin, Stanislav; Nguyen, Hien; Kienzler, Andrea; Clima, Lilia; Bannwarth, Willi; Appel, Bettina; Müller, Sabine; Seidel, Claus A M

    2011-03-02

    In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A

  1. Pivotal role of extended linker 2 in the activation of Gα by G protein-coupled receptor.

    Science.gov (United States)

    Huang, Jianyun; Sun, Yutong; Zhang, J Jillian; Huang, Xin-Yun

    2015-01-02

    G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Solid-phase synthesis of polyfunctional polylysine dendrons using aldehyde linkers

    DEFF Research Database (Denmark)

    Svenssen, Daniel K.; Mirsharghi, Sahar; Boas, Ulrik

    2014-01-01

    A straightforward method for the solid-phase synthesis of C-terminally modified polylysine dendrons has been developed by applying bisalkoxybenzaldehyde and trisalkoxybenzaldehyde linkers. The method has been used for the synthesis of polylysine dendrons with a variety of C-terminal ‘tail groups’...

  3. Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

    Science.gov (United States)

    Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

    2016-03-02

    Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

  4. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

    Science.gov (United States)

    Morin, Emmanuelle; Kohler, Annegret; Baker, Adam R.; Foulongne-Oriol, Marie; Lombard, Vincent; Nagye, Laszlo G.; Ohm, Robin A.; Patyshakuliyeva, Aleksandrina; Brun, Annick; Aerts, Andrea L.; Bailey, Andrew M.; Billette, Christophe; Coutinho, Pedro M.; Deakin, Greg; Doddapaneni, Harshavardhan; Floudas, Dimitrios; Grimwood, Jane; Hildén, Kristiina; Kües, Ursula; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Murat, Claude; Riley, Robert W.; Salamov, Asaf A.; Schmutz, Jeremy; Subramanian, Venkataramanan; Wösten, Han A. B.; Xu, Jianping; Eastwood, Daniel C.; Foster, Gary D.; Sonnenberg, Anton S. M.; Cullen, Dan; de Vries, Ronald P.; Lundell, Taina; Hibbett, David S.; Henrissat, Bernard; Burton, Kerry S.; Kerrigan, Richard W.; Challen, Michael P.; Grigoriev, Igor V.; Martin, Francis

    2012-01-01

    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics. PMID:23045686

  5. Linker histones: novel insights into structure-specific recognition of the nucleosome.

    Science.gov (United States)

    Cutter, Amber R; Hayes, Jeffrey J

    2017-04-01

    Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing the wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures, influencing nucleosome spacing on DNA, and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while highlighting the remaining questions.

  6. A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

    DEFF Research Database (Denmark)

    1997-01-01

    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide......-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent...

  7. 5-fold increase of hydrogen uptake in MOF74 through linker decorations

    Science.gov (United States)

    Thonhauser, T.; Zuluaga, S.; Harrison, D.; Welchman, E.; Arter, C.

    We present ab initio results for linker decorations in Mg-MOF74-i.e. attaching various metals  = Li, Na, K, Sc, Cr, Mn, Fe, Ni, Cu, Zn, Rb, Pd, Ag, and Pt near the ring of the linker-creating new strong adsorption sites and thus maximizing small molecule uptake. We find that in most cases these decorations influence the overall form and structure of Mg-MOF74 only marginally. After the initial screening we chose metals that bind favorably to the linker and further investigate adsorption of H2, CO2, and H2O for  = Li, Na, K, and Sc. For the case of H2 we show that up to 24 additional guest molecules can be adsorbed in the MOF unit cell, with binding energies comparable to the original open-metal sites at the six corners of the channel. This leads to a 5-fold increase of the molecule uptake in Mg-MOF74, with tremendous impact on many applications in general and hydrogen storage in particular-where the gravimetric hydrogen density increases from 1 . 63 mass% to 7 . 28 mass% and the volumetric density from 15.10 g H2 L-1 to 75.50 g H2 L-1. This work was supported by NSF Grant No. DMR-1145968.

  8. Temperature-triggered release of a liquid cross-linker micro-encapsulated in a glassy polymer for low temperature curing

    NARCIS (Netherlands)

    Senatore, D.; Cate, ten A.T.; Laven, J.; Benthem, van R.A.T.M.; With, de G.

    2013-01-01

    In order to prevent a liquid epoxy cross-linker from premature, Arrhenius-law predicted, reaction with an acid-functional polyester resin, the liquid cross-linker has been physically separated from the resin by encapsulation while release is only possible by a temperature-controlled trigger. The

  9. Epigenetics and autism spectrum disorder: A report of an autism case with mutation in H1 linker histone HIST1H1e and literature review.

    Science.gov (United States)

    Duffney, Lara J; Valdez, Purnima; Tremblay, Martine W; Cao, Xinyu; Montgomery, Sarah; McConkie-Rosell, Allyn; Jiang, Yong-Hui

    2018-04-27

    Genetic mutations in genes encoding proteins involved in epigenetic machinery have been reported in individuals with autism spectrum disorder (ASD), intellectual disability, congenital heart disease, and other disorders. H1 histone linker protein, the basic component in nucleosome packaging and chromatin organization, has not been implicated in human disease until recently. We report a de novo deleterious mutation of histone cluster 1 H1 family member e (HIST1H1E; c.435dupC; p.Thr146Hisfs*50), encoding H1 histone linker protein H1.4, in a 10-year-old boy with autism and intellectual disability diagnosed through clinical whole exome sequencing. The c.435dupC at the 3' end of the mRNA leads to a frameshift and truncation of the positive charge in the carboxy-terminus of the protein. An expression study demonstrates the mutation leads to reduced protein expression, supporting haploinsufficiency of HIST1H1E protein and loss of function as an underlying mechanism of dysfunction in the brain. Taken together with other recent cases with mutations of HIST1H1E in intellectual disability, the evidence supporting the link to causality in disease is strong. Our finding implicates the deficiency of H1 linker histone protein in autism. The systematic review of candidate genes implicated in ASD revealed that 42 of 215 (19.5%) genes are directly involved in epigenetic regulations and the majority of these genes belong to histone writers, readers, and erasers. While the mechanism of how haploinsufficiency of HIST1H1E causes autism is entirely unknown, our report underscores the importance of further study of the function of this protein and other histone linker proteins in brain development. © 2018 Wiley Periodicals, Inc.

  10. Characterization and sequence analysis of cysteine and glycine-rich ...

    African Journals Online (AJOL)

    Primers specific for CSRP3 were designed using known cDNA sequences of Bos taurus published in database with different accession numbers. Polymerase chain reaction (PCR) was performed and products were purified and sequenced. Sequence analysis and alignment were carried out using CLUSTAL W (1.83).

  11. Extremely stretchable thermosensitive hydrogels by introducing slide-ring polyrotaxane cross-linkers and ionic groups into the polymer network

    Science.gov (United States)

    Bin Imran, Abu; Esaki, Kenta; Gotoh, Hiroaki; Seki, Takahiro; Ito, Kohzo; Sakai, Yasuhiro; Takeoka, Yukikazu

    2014-01-01

    Stimuli-sensitive hydrogels changing their volumes and shapes in response to various stimulations have potential applications in multiple fields. However, these hydrogels have not yet been commercialized due to some problems that need to be overcome. One of the most significant problems is that conventional stimuli-sensitive hydrogels are usually brittle. Here we prepare extremely stretchable thermosensitive hydrogels with good toughness by using polyrotaxane derivatives composed of α-cyclodextrin and polyethylene glycol as cross-linkers and introducing ionic groups into the polymer network. The ionic groups help the polyrotaxane cross-linkers to become well extended in the polymer network. The resulting hydrogels are surprisingly stretchable and tough because the cross-linked α-cyclodextrin molecules can move along the polyethylene glycol chains. In addition, the polyrotaxane cross-linkers can be used with a variety of vinyl monomers; the mechanical properties of the wide variety of polymer gels can be improved by using these cross-linkers. PMID:25296246

  12. Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

    Science.gov (United States)

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity. PMID:25029439

  13. Saccharomyces cerevisiae Linker Histone—Hho1p Maintains Chromatin Loop Organization during Ageing

    Directory of Open Access Journals (Sweden)

    Katya Uzunova

    2013-01-01

    Full Text Available Intricate, dynamic, and absolutely unavoidable ageing affects cells and organisms through their entire lifetime. Driven by diverse mechanisms all leading to compromised cellular functions and finally to death, this process is a challenge for researchers. The molecular mechanisms, the general rules that it follows, and the complex interplay at a molecular and cellular level are yet little understood. Here, we present our results showing a connection between the linker histones, the higher-order chromatin structures, and the process of chronological lifespan of yeast cells. By deleting the gene for the linker histone in Saccharomyces cerevisiae we have created a model for studying the role of chromatin structures mainly at its most elusive and so far barely understood higher-order levels of compaction in the processes of yeast chronological lifespan. The mutant cells demonstrated controversial features showing slower growth than the wild type combined with better survival during the whole process. The analysis of the global chromatin organization during different time points demonstrated certain loss of the upper levels of chromatin compaction in the cells without linker histone. The results underlay the importance of this histone for the maintenance of the chromatin loop structures during ageing.

  14. Impact of cathepsin B-sensitive triggers and hydrophilic linkers on in vitro efficacy of novel site-specific antibody-drug conjugates.

    Science.gov (United States)

    Bryden, Francesca; Martin, Camille; Letast, Stéphanie; Lles, Eva; Viéitez-Villemin, Inmaculada; Rousseau, Anaïs; Colas, Cyril; Brachet-Botineau, Marie; Allard-Vannier, Emilie; Larbouret, Christel; Viaud-Massuard, Marie-Claude; Joubert, Nicolas

    2018-03-14

    Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG 12 spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.

  15. Bifunctional bridging linker-assisted synthesis and characterization of TiO{sub 2}/Au nanocomposites

    Energy Technology Data Exchange (ETDEWEB)

    Žunič, Vojka, E-mail: vojka.zunic@ijs.si, E-mail: vojka13@gmail.com; Kurtjak, Mario; Suvorov, Danilo [Jožef Stefan Institute, Advanced Materials Department (Slovenia)

    2016-11-15

    Using a simple organic bifunctional bridging linker, titanium dioxide (TiO{sub 2}) nanoparticles were coupled with the Au nanoparticles to form TiO{sub 2}/Au nanocomposites with a variety of Au loadings. This organic bifunctional linker, meso-2,3-dimercaptosuccinic acid, contains two types of functional groups: (i) the carboxyl group, which enables binding to the TiO{sub 2}, and (ii) the thiol group, which enables binding to the Au. In addition, the organic bifunctional linker acts as a stabilizing agent to prevent the agglomeration and growth of the Au particles, resulting in the formation of highly dispersed Au nanoparticles. To form the TiO{sub 2}/Au nanocomposites in a simple way, we deliberately applied a synthetic method that simultaneously ensures: (i) the capping of the Au nanoparticles and (ii) the binding of different amounts of Au to the TiO{sub 2}. The TiO{sub 2}/Au nanocomposites formed with this method show enhanced UV and Vis photocatalytic activities when compared to the pure TiO{sub 2} nanopowders.Graphical Abstract.

  16. Release of 3-methyladenine from linker and core DNA of chromatin by a purified DNA glycosylase

    International Nuclear Information System (INIS)

    Heller, E.P.; Goldthwait, D.A.

    1983-01-01

    Oligonucleosomes were isolated from [ 14 C]thymidine-labeled HeLa cells by digestion of the nuclei with micrococcal nuclease and were then alkylated with [ 3 H]methylnitrosourea. Nucleosome core particles were also prepared by further digestion of the oligonucleosomes. The distribution of 3 H-labeled methyl groups in the linker versus the core DNA was established by a determination of 3 H: 14 C ratios in oligonucleosome and core DNA. The ratios in the core DNA of 145 and 165 base pair DNA fragments were 5.2 and 5.4, respectively, while the ratio in the oligonucleosomal DNA was 8.2. Assuming an equal mixture (as determined) of 145 and 165 base pair fragments of DNA in the 185 base pair repeat, the relative concentration of 3 H methyl groups in the linker versus the core DNA was 4.2. Thus, 45% of the 3 H methyl groups were in the linker DNA, and 55% were in the core DNA. Some shielding of the DNA was evident during alkylation. The concentrations of alkyl groups on the linker and core DNA were 67 and 12% of that found on free DNA alkylated under comparable conditions. No evidence for preferential shielding of the major or minor groove was observed. The purified 3-methyladenine DNA glycosylase I of Escherichia coli released approximately 37% of the 3-methyladenine from the linker DNA and 13% from the core DNA. The limited enzymatic removal of 3-methyladenine in vitro compared to the efficient removal in vivo suggests that conformational changes of the oligonucleosome and core structure must occur for total repair

  17. Effects of metal-rich particulate matter exposure on exogenous and endogenous viral sequence methylation in healthy steel-workers.

    Science.gov (United States)

    Mercorio, Roberta; Bonzini, Matteo; Angelici, Laura; Iodice, Simona; Delbue, Serena; Mariani, Jacopo; Apostoli, Pietro; Pesatori, Angela Cecilia; Bollati, Valentina

    2017-11-01

    Inhaled particles have been shown to produce systemic changes in DNA methylation. Global hypomethylation has been associated to viral sequence reactivation, possibly linked to the activation of pro-inflammatory pathways occurring after exposure. This observation provides a rationale to investigate viral sequence (both exogenous and endogenous) methylation in association to metal-rich particulate matter exposure. To verify this hypothesis, we chose the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), respectively as a paradigm of an exogenous and an endogenous retroviral sequence, to be investigated by bisulfite PCR Pyrosequencing. We enrolled 63 male workers in an electric furnace steel plant, exposed to high level of metal-rich particulate matter. Comparing samples obtained in the first day of a work week (time 0-baseline, after 2 days off work) and the samples obtained after 3 days of work (time 1-post exposure), the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (mean baseline = 56.7%5mC; mean post-exposure = 47.9%5mC; p-value = 0.009), whereas the mean methylation of HERV-w did not significantly differ. Individual exposure to inhalable particles and metals was estimated based on measures in all working areas and time spent by the study subjects in each area. In a regression model adjusted for age, body mass index and smoking, PM and metal components had a positive association with EBV-Wp methylation (i.e. PM10: β = 5.99, p-value < 0.038; nickel: β = 17.82, p-value = 0.02; arsenic: β = 13.59, p-value < 0.015). The difference observed comparing baseline and post-exposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and PM/metal exposure may represent a more stable adaptive mechanism. Future studies investigating a larger panel of viral sequences could better elucidate

  18. Iminodiacetic acid as bifunctional linker for dimerization of cyclic RGD peptides

    International Nuclear Information System (INIS)

    Xu, Dong; Zhao, Zuo-Quan; Chen, Shu-Ting; Yang, Yong; Fang, Wei; Liu, Shuang

    2017-01-01

    Introduction: In this study, I2P-RGD 2 was used as the example to illustrate a novel approach for dimerization of cyclic RGD peptides. The main objective of this study was to explore the impact of bifunctional linkers (glutamic acid vs. iminodiacetic acid) on tumor-targeting capability and excretion kinetics of the 99m Tc-labeled dimeric cyclic RGD peptides. Methods: HYNIC-I2P-RGD 2 was prepared by reacting I2P-RGD 2 with HYNIC-OSu in the presence of diisopropylethylamine, and was evaluated for its α v β 3 binding affinity against 125 I-echistatin bound to U87MG glioma cells. 99m Tc-I2P-RGD 2 was prepared with high specific activity (~185 GBq/μmol). The athymic nude mice bearing U87MG glioma xenografts were used to evaluate its biodistribution properties and image quality in comparison with those of 99m Tc-3P-RGD 2 . Results: The IC 50 value for HYNIC-I2P-RGD 2 was determined to be 39 ± 6 nM, which was very close to that (IC 50 = 33 ± 5 nM) of HYNIC-3P-RGD 2 . Replacing glutamic acid with iminodiacetic acid had little impact on α v β 3 binding affinity of cyclic RGD peptides. 99m Tc-I2P-RGD 2 and 99m Tc-3P-RGD 2 shared similar tumor uptake values over the 2 h period, and its α v β 3 -specificity was demonstrated by a blocking experiment. The uptake of 99m Tc-I2P-RGD 2 was significantly lower than 99m Tc-3P-RGD 2 in the liver and kidneys. The U87MG glioma tumors were visualized by SPECT with excellent contrast using both 99m Tc-I2P-RGD 2 and 99m Tc-3P-RGD 2 . Conclusion: Iminodiacetic acid is an excellent bifunctional linker for dimerization of cyclic RGD peptides. Bifunctional linkers have significant impact on the excretion kinetics of 99m Tc radiotracers. Because of its lower liver uptake and better tumor/liver ratios, 99m Tc-I2P-RGD 2 may have advantages over 99m Tc-3P-RGD 2 for diagnosis of tumors in chest region. -- Graphical abstract: This report presents novel approach for dimerization of cyclic RGD peptides using iminodiacetic acid as a

  19. The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating.

    Directory of Open Access Journals (Sweden)

    Chai Ann Ng

    Full Text Available Human ether-à-go-go-related gene (hERG K(+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.

  20. Onset of grain filling is associated with a change in properties of linker histone variants in maize kernels

    DEFF Research Database (Denmark)

    Kalamajka, R.; Finnie, Christine; Grasser, K.D.

    2010-01-01

    ) initiation of storage synthesis. Six linker histone gene products were identified by MALDI-TOF mass spectrometry. A marked shift of around 4 pH units was observed for the linker histone spot pattern after 2D-gel electrophoresis when comparing the proteins of 11 and 16 dap kernels. The shift from acidic...

  1. Synthesis of two new alkyne-bearing linkers used for the preparation of siRNA for labeling by click chemistry with fluorine-18

    International Nuclear Information System (INIS)

    Flagothier, Jessica; Kaisin, Geoffroy; Mercier, Frederic; Thonon, David; Teller, Nathalie; Wouters, Johan; Luxen, André

    2012-01-01

    Oligonucleotides (ONs) and more particularly siRNAs are promising drugs but their pharmacokinetics and biodistribution are widely unknown. Positron Emission Tomography (PET) using fluorine-18 is a suitable technique to quantify these biological processes. Click chemistry (Huisgen cycloaddition) is the current method for labeling siRNA. In order to study the influence of a linker bearing by [ 18 F] labeled ONs, on the in vivo pharmacokinetic and metabolism, we have developed two modified ONs by two new linkers. Here we report the synthesis of two alkyne-bearing linkers, the incorporation onto a ONs and the conjugation by click chemistry with a [ 18 F] prosthetic group. - Highlights: ► Synthesis of two new alkyne linkers. ► Functionalization at the 3′-end siRNA by alkyne linker derived of proline. ► Click chemistry between alkyne modified siRNA and [ 18 F] prosthetic group.

  2. Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

    Science.gov (United States)

    Shinohara, Ken-ichi; Bando, Toshikazu; Sasaki, Shunta; Sakakibara, Yogo; Minoshima, Masafumi; Sugiyama, Hiroshi

    2006-03-01

    DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (Ralkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

  3. Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths

    International Nuclear Information System (INIS)

    Noach, Ilit; Alber, Orly; Bayer, Edward A.; Lamed, Raphael; Levy-Assaraf, Maly; Shimon, Linda J. W.; Frolow, Felix

    2007-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray characterization of two protein constructs of the second type II cohesin module from A. cellulolyticus ScaB are described. Both constructs contain the native N-terminal linker, but only one of them contains the full-length 45-residue C-terminal linker; the other contains a five-residue segment of this linker. The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2-S) and the second incorporating the full native 45-residue linker (CohB2-L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2-S and CohB2-L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2 1 2 1 2 1 with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 Å for CohB2-S and space group P2 1 2 1 2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2-L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2-S contains three cohesin molecules, while that of CohB2-L contains two molecules

  4. POST-MERGER SIGNATURES OF RED-SEQUENCE GALAXIES IN RICH ABELL CLUSTERS AT z ∼< 0.1

    International Nuclear Information System (INIS)

    Sheen, Yun-Kyeong; Yi, Sukyoung K.; Lee, Jaehyun; Ree, Chang H.

    2012-01-01

    We have investigated the post-merger signatures of red-sequence galaxies in rich Abell clusters at z ∼ r < –20) cluster red-sequence galaxies show post-merger signatures in four clusters consistently. Most (∼71%) of the featured galaxies were found to be bulge dominated, and for the subsample of bulge-dominated red-sequence galaxies, the post-merger fraction rises to ∼38%. We also found that roughly 4% of bulge-dominated red-sequence galaxies interact (ongoing merger). A total of 42% (38% post-merger, 4% ongoing merger) of galaxies show merger-related features. Compared to a field galaxy study with a similar limiting magnitude by van Dokkum in 2005, our cluster study presents a similar post-merger fraction but a markedly lower ongoing merger fraction. The merger fraction derived is surprisingly high for the high density of our clusters, where the fast internal motions of galaxies are thought to play a negative role in galaxy mergers. The fraction of post-merger and ongoing merger galaxies can be explained as follows. Most of the post-merger galaxies may have carried over their merger features from their previous halo environment, whereas interacting galaxies interact in the current cluster in situ. According to our semi-analytic calculation, massive cluster halos may very well have experienced tens of halo mergers over the last 4-5 Gyr; post-merger features last that long, allowing these features to be detected in our clusters today. The apparent lack of dependence of the merger fraction on the clustocentric distance is naturally explained this way. In this scenario, the galaxy morphology and properties can be properly interpreted only when the halo evolution characteristics are understood first.

  5. CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

    Science.gov (United States)

    Hem, C D; Ekornhol, M; Granum, S; Sundvold-Gjerstad, V; Spurkland, A

    2017-02-01

    The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr) 629 and linker of activated T cells (LAT) pTyr 171 , pTyr 191 and pTyr 226 . © 2016 The Foundation for the Scandinavian Journal of Immunology.

  6. Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

    Science.gov (United States)

    Zheng, Yanhua; Lu, Zhimin

    2013-01-01

    Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

  7. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins......, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA...

  8. Germline-specific H1 variants: the "sexy" linker histones.

    Science.gov (United States)

    Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

    2016-03-01

    The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties.

  9. A Linker for the Solid-Phase Synthesis of Hydroxamic Acids and Identification of HDAC6 Inhibitors

    DEFF Research Database (Denmark)

    Bang, Claus Gunnar; Jensen, Jakob Feldthusen; Cohrt, Anders Emil O'Hanlon

    2017-01-01

    We herein present broadly useful, readily available and nonintegral hydroxylamine linkers for the routine solid-phase synthesis of hydroxamic acids. The developed protocols enable the efficient synthesis and release of a wide range of hydroxamic acids from various resins, relying on high control...... and flexibility with respect to reagents and synthetic processes. A trityl-based hydroxylamine linker was used to synthesize a library of peptide hydroxamic acids. The inhibitory effects of the compounds were examined for seven HDAC enzyme subtypes using a chemiluminescence-based assay....

  10. Desorption of 3,3′-diindolylmethane from imprinted particles: An impact of cross-linker structure on binding capacity and selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Klejn, Dorota; Luliński, Piotr; Maciejewska, Dorota, E-mail: dorota.maciejewska@wum.edu.pl

    2015-11-01

    Here, seven cross-linkers (six polar diacrylates or dimethacrylates of different lengths between double bonds, and one aromatic-divinylbenzene) were used to examine the impact of the cross-linker on binding capacity and selectivity of 3,3′-diindolylmethane (DIM) imprinted material. DIM participates in the suppression of viability of human ovarian and human breast cancer cell lines, but has low bioavailability. The investigations of novel imprinted polymer matrices for improvement of DIM release could allow to utilize not only a potency of DIM but also similar alkaloids, which are the important compounds with pharmacological activity. The bulk, thermal radical copolymerization of the cross-linkers in the presence of 3,3′-diindolylmethane (the template) and allylamine (the functional monomer) in dimethyl sulfoxide or in carbon tetrachloride (porogens) was carried out. The binding capacities of imprinted and non-imprinted polymers were compared, and two polymers (these were prepared using ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate as the cross-linkers) with the highest selectivity and binding capacity were selected to desorption test. The desorption profile of polymer prepared using polyethylene glycol dimethacrylate as the cross-linker revealed sustained release of 3,3′-diindolylmethane, and this system was selected for further optimization of the cross-linker amounts. The morphology and structure of the selected particles were analyzed using SEM micrographs, {sup 13}C CP/MAS NMR spectroscopy, and BET measurements. The desorption of 3,3′-diindolylmethane from poly(allylamine-co-polyethylene glycol dimethacrylate) particles was in accordance with pseudo-second-order kinetics and the simplified Higuchi model indicated the diffusion controlled release of 3,3′-diindolylmethane. - Graphical abstract: Sustained release of 3,3′-diindolylmethane from cavity in imprinted poly(allylamine-co-polyethylene glycol dimethacrylate

  11. Desorption of 3,3′-diindolylmethane from imprinted particles: An impact of cross-linker structure on binding capacity and selectivity

    International Nuclear Information System (INIS)

    Klejn, Dorota; Luliński, Piotr; Maciejewska, Dorota

    2015-01-01

    Here, seven cross-linkers (six polar diacrylates or dimethacrylates of different lengths between double bonds, and one aromatic-divinylbenzene) were used to examine the impact of the cross-linker on binding capacity and selectivity of 3,3′-diindolylmethane (DIM) imprinted material. DIM participates in the suppression of viability of human ovarian and human breast cancer cell lines, but has low bioavailability. The investigations of novel imprinted polymer matrices for improvement of DIM release could allow to utilize not only a potency of DIM but also similar alkaloids, which are the important compounds with pharmacological activity. The bulk, thermal radical copolymerization of the cross-linkers in the presence of 3,3′-diindolylmethane (the template) and allylamine (the functional monomer) in dimethyl sulfoxide or in carbon tetrachloride (porogens) was carried out. The binding capacities of imprinted and non-imprinted polymers were compared, and two polymers (these were prepared using ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate as the cross-linkers) with the highest selectivity and binding capacity were selected to desorption test. The desorption profile of polymer prepared using polyethylene glycol dimethacrylate as the cross-linker revealed sustained release of 3,3′-diindolylmethane, and this system was selected for further optimization of the cross-linker amounts. The morphology and structure of the selected particles were analyzed using SEM micrographs, 13 C CP/MAS NMR spectroscopy, and BET measurements. The desorption of 3,3′-diindolylmethane from poly(allylamine-co-polyethylene glycol dimethacrylate) particles was in accordance with pseudo-second-order kinetics and the simplified Higuchi model indicated the diffusion controlled release of 3,3′-diindolylmethane. - Graphical abstract: Sustained release of 3,3′-diindolylmethane from cavity in imprinted poly(allylamine-co-polyethylene glycol dimethacrylate). - Highlights:

  12. High-resolution two-dimensional liquid chromatography analysis of key linker drug intermediate used in antibody drug conjugates.

    Science.gov (United States)

    Venkatramani, C J; Huang, Shu Rong; Al-Sayah, Mohammad; Patel, Ila; Wigman, Larry

    2017-10-27

    In this manuscript, the application of high-resolution sampling (HRS) two-dimensional liquid chromatography (2D-LC) in the detailed analysis of key linker drug intermediate is presented. Using HRS, selected regions of the primary column eluent were transferred to a secondary column with fidelity enabling qualitative and quantitative analysis of linker drugs. The primary column purity of linker drug intermediate ranged from 88.9% to 94.5% and the secondary column purity ranged from 99.6% to 99.9%, showing lot-to-lot variability, significant differences between the three lots, and substantiating the synthetic and analytical challenges of ADCs. Over 15 impurities co-eluting with the linker drug intermediate in the primary dimension were resolved in the secondary dimension. The concentrations of most of these impurities were over three orders of magnitude lower than the linker drug. Effective peak focusing and high-speed secondary column analysis resulted in sharp peaks in the secondary dimension, improving the signal-to-noise ratios. The sensitivity of 2D-LC separation was over five fold better than conventional HPLC separation. The limit of quantitation (LOQ) was less than 0.01%. Many peaks originating from primary dimension were resolved into multiple components in the complementary secondary dimension, demonstrating the complexity of these samples. The 2D-LC was highly reproducible, showing good precision between runs with%RSD of peak areas less than 0.1 for the main component. The absolute difference in the peak areas of impurities less than 0.1% were within ±0.01% and for impurities in the range of 0.1%-0.3%, the absolute difference were ±0.02%, which are comparable to 1D-LC. The overall purity of the linker drug intermediate was determined from the product of primary and secondary column purity (HPLC Purity=%peak area of main component in the primary dimension×%peak area of main component in the secondary dimension). Additionally, the 2D-LC separation enables

  13. Short sequence motifs, overrepresented in mammalian conservednon-coding sequences

    Energy Technology Data Exchange (ETDEWEB)

    Minovitsky, Simon; Stegmaier, Philip; Kel, Alexander; Kondrashov,Alexey S.; Dubchak, Inna

    2007-02-21

    Background: A substantial fraction of non-coding DNAsequences of multicellular eukaryotes is under selective constraint. Inparticular, ~;5 percent of the human genome consists of conservednon-coding sequences (CNSs). CNSs differ from other genomic sequences intheir nucleotide composition and must play important functional roles,which mostly remain obscure.Results: We investigated relative abundancesof short sequence motifs in all human CNSs present in the human/mousewhole-genome alignments vs. three background sets of sequences: (i)weakly conserved or unconserved non-coding sequences (non-CNSs); (ii)near-promoter sequences (located between nucleotides -500 and -1500,relative to a start of transcription); and (iii) random sequences withthe same nucleotide composition as that of CNSs. When compared tonon-CNSs and near-promoter sequences, CNSs possess an excess of AT-richmotifs, often containing runs of identical nucleotides. In contrast, whencompared to random sequences, CNSs contain an excess of GC-rich motifswhich, however, lack CpG dinucleotides. Thus, abundance of short sequencemotifs in human CNSs, taken as a whole, is mostly determined by theiroverall compositional properties and not by overrepresentation of anyspecific short motifs. These properties are: (i) high AT-content of CNSs,(ii) a tendency, probably due to context-dependent mutation, of A's andT's to clump, (iii) presence of short GC-rich regions, and (iv) avoidanceof CpG contexts, due to their hypermutability. Only a small number ofshort motifs, overrepresented in all human CNSs are similar to bindingsites of transcription factors from the FOX family.Conclusion: Human CNSsas a whole appear to be too broad a class of sequences to possess strongfootprints of any short sequence-specific functions. Such footprintsshould be studied at the level of functional subclasses of CNSs, such asthose which flank genes with a particular pattern of expression. Overallproperties of CNSs are affected by

  14. Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yuan Tong

    2010-01-01

    Full Text Available Abstract Background Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs, representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. Results As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC at Ohio State University for full accessibility by the Arabidopsis research community. Conclusions Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

  15. Investigation of the Linker Swing Motion in the Zeolitic Imidazolate Framework ZIF-90

    KAUST Repository

    Zheng, Bin; Fu, Fang; Wang, Lian Li; Yang, Limin; Zhu, Yihan; Du, Huiling

    2018-01-01

    The linker swing motion in the zeolitic imidazolate framework ZIF-90 is investigated by density functional theory (DFT) calculation, molecular dynamics (MD) and grand-canonical Monte Carlo (GCMC) simulations. The relation between the terminal

  16. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle.

    Science.gov (United States)

    Hergeth, Sonja P; Schneider, Robert

    2015-11-01

    The linker histone H1 family members are a key component of chromatin and bind to the nucleosomal core particle around the DNA entry and exit sites. H1 can stabilize both nucleosome structure and higher-order chromatin architecture. In general, H1 molecules consist of a central globular domain with more flexible tail regions at both their N- and C-terminal ends. The existence of multiple H1 subtypes and a large variety of posttranslational modifications brings about a considerable degree of complexity and makes studying this protein family challenging. Here, we review recent progress in understanding the function of linker histones and their subtypes beyond their role as merely structural chromatin components. We summarize current findings on the role of H1 in heterochromatin formation, transcriptional regulation and embryogenesis with a focus on H1 subtypes and their specific modifications. © 2015 The Authors.

  17. Efficient Naphthalenediimide-Based Hole Semiconducting Polymer with Vinylene Linkers between Donor and Acceptor Units

    KAUST Repository

    Zhang, Lei

    2016-11-04

    We demonstrate a new method to reverse the polarity and charge transport behavior of naphthalenediimide (NDI)-based copolymers by inserting a vinylene linker between the donor and acceptor units. The vinylene linkers minimize the intrinsic steric congestion between the NDI and thiophene moieties to prompt backbone planarity. The polymers with vinylene linkers exhibit electron n-channel transport characteristics under vacuum, similar to the benchmark polymer, P(NDI2OD-T2). To our surprise, when the polymers are measured in air, the dominant carrier type switches from n- to p-type and yield hole mobilities up to 0.45 cm(2) s(-1) with hole to electron mobility ratio of three (mu(h)/mu(e), similar to 3), which indicates that the hole density in the active layer can be significantly increased by exposure to air. This increase is consistent with the intrinsic more delocalized nature of the highest occupied molecular orbital of the charged vinylene polymer, as estimated by density functional theory (DFT) calculations, which facilitates hole transport within the polymer chains. This is the first demonstration of an efficient NDI-based hole semiconducting polymer, which will enable new developments in all-polymer solar cells, complementary circuits, and dopable polymers for use in thermoelectrics.

  18. Efficient Naphthalenediimide-Based Hole Semiconducting Polymer with Vinylene Linkers between Donor and Acceptor Units

    KAUST Repository

    Zhang, Lei; Rose, Bradley Daniel; Liu, Yao; Nahid, Masrur M.; Gann, Eliot; Ly, Jack; Zhao, Wei; Rosa, Stephen J.; Russell, Thomas P.; Facchetti, Antonio; McNei, Christopher R.; Bredas, Jean-Luc; Briseno, Alejandro L.

    2016-01-01

    We demonstrate a new method to reverse the polarity and charge transport behavior of naphthalenediimide (NDI)-based copolymers by inserting a vinylene linker between the donor and acceptor units. The vinylene linkers minimize the intrinsic steric congestion between the NDI and thiophene moieties to prompt backbone planarity. The polymers with vinylene linkers exhibit electron n-channel transport characteristics under vacuum, similar to the benchmark polymer, P(NDI2OD-T2). To our surprise, when the polymers are measured in air, the dominant carrier type switches from n- to p-type and yield hole mobilities up to 0.45 cm(2) s(-1) with hole to electron mobility ratio of three (mu(h)/mu(e), similar to 3), which indicates that the hole density in the active layer can be significantly increased by exposure to air. This increase is consistent with the intrinsic more delocalized nature of the highest occupied molecular orbital of the charged vinylene polymer, as estimated by density functional theory (DFT) calculations, which facilitates hole transport within the polymer chains. This is the first demonstration of an efficient NDI-based hole semiconducting polymer, which will enable new developments in all-polymer solar cells, complementary circuits, and dopable polymers for use in thermoelectrics.

  19. Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains.

    Science.gov (United States)

    Lindfors, Hanna E; Venkata, Bharat Somireddy; Drijfhout, Jan W; Ubbink, Marcellus

    2011-02-18

    The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Preparation of value-added metal-organic frameworks (MOFs) using waste PET bottles as source of acid linker

    CSIR Research Space (South Africa)

    Dyosiba, Xoliswa

    2016-12-01

    Full Text Available of Value-added Metal-organic Frameworks (MOFs) Using Waste PET Bottles as Source of Acid Linker Xoliswa Dyosiba, Jianwei Ren, Nicholas M. Musyoka, Henrietta W. Langmi, Mkhulu Mathe, Maurice S. Onyango PII: S2214-9937(16)30053-7 DOI: doi:10.1016/j..., Hen- rietta W. Langmi, Mkhulu Mathe, Maurice S. Onyango, Preparation of Value-added Metal-organic Frameworks (MOFs) Using Waste PET Bottles as Source of Acid Linker, Sustainable Materials and Technologies (2016), doi:10.1016/j.susmat.2016...

  1. Classification of G-protein coupled receptors based on a rich generation of convolutional neural network, N-gram transformation and multiple sequence alignments.

    Science.gov (United States)

    Li, Man; Ling, Cheng; Xu, Qi; Gao, Jingyang

    2018-02-01

    Sequence classification is crucial in predicting the function of newly discovered sequences. In recent years, the prediction of the incremental large-scale and diversity of sequences has heavily relied on the involvement of machine-learning algorithms. To improve prediction accuracy, these algorithms must confront the key challenge of extracting valuable features. In this work, we propose a feature-enhanced protein classification approach, considering the rich generation of multiple sequence alignment algorithms, N-gram probabilistic language model and the deep learning technique. The essence behind the proposed method is that if each group of sequences can be represented by one feature sequence, composed of homologous sites, there should be less loss when the sequence is rebuilt, when a more relevant sequence is added to the group. On the basis of this consideration, the prediction becomes whether a query sequence belonging to a group of sequences can be transferred to calculate the probability that the new feature sequence evolves from the original one. The proposed work focuses on the hierarchical classification of G-protein Coupled Receptors (GPCRs), which begins by extracting the feature sequences from the multiple sequence alignment results of the GPCRs sub-subfamilies. The N-gram model is then applied to construct the input vectors. Finally, these vectors are imported into a convolutional neural network to make a prediction. The experimental results elucidate that the proposed method provides significant performance improvements. The classification error rate of the proposed method is reduced by at least 4.67% (family level I) and 5.75% (family Level II), in comparison with the current state-of-the-art methods. The implementation program of the proposed work is freely available at: https://github.com/alanFchina/CNN .

  2. A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

    DEFF Research Database (Denmark)

    Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano

    2016-01-01

    with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively...

  3. Distribution of linker histone variants during plant cell differentiation in the developmental zones of the maize root, dedifferentiation in callus culture after auxin treatment

    Directory of Open Access Journals (Sweden)

    ANASTASIOS ALATZAS

    2008-01-01

    Full Text Available Although several linker histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study, we identified linker histone variants by using specific anti-histone Hl antibodies. Each variant's ratio to total Hl in the three developmental zones of maize (Zea mays L. root and in callus cultures derived from them was estimated in order to define possible alterations either during plant cell differentiation or during their dedifferentiation. We also evaluated linker histone variants' ratios in the developmental zones of maize roots treated with auxin in order to examine the effects of exogenous applied auxin to linker histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing each variant and correlate them with the physiological status of the plant cells. According to the results presented in this study, linker histone variants' ratios are altered in the developmental zones of maize root, while they are similar to the meristematic zone in samples from callus cultures and to the differentiation zone in samples from roots treated with auxin. We propose that the alterations in linker histone variants' ratios are correlated with plant cell differentiation and dedifferentiation.

  4. Mutations in B3GALT6, which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders.

    Science.gov (United States)

    Nakajima, Masahiro; Mizumoto, Shuji; Miyake, Noriko; Kogawa, Ryo; Iida, Aritoshi; Ito, Hironori; Kitoh, Hiroshi; Hirayama, Aya; Mitsubuchi, Hiroshi; Miyazaki, Osamu; Kosaki, Rika; Horikawa, Reiko; Lai, Angeline; Mendoza-Londono, Roberto; Dupuis, Lucie; Chitayat, David; Howard, Andrew; Leal, Gabriela F; Cavalcanti, Denise; Tsurusaki, Yoshinori; Saitsu, Hirotomo; Watanabe, Shigehiko; Lausch, Ekkehart; Unger, Sheila; Bonafé, Luisa; Ohashi, Hirofumi; Superti-Furga, Andrea; Matsumoto, Naomichi; Sugahara, Kazuyuki; Nishimura, Gen; Ikegawa, Shiro

    2013-06-06

    Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  5. Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2015-01-01

    Full Text Available Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

  6. SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology

    DEFF Research Database (Denmark)

    Kim, Se Hyeuk; Cavaleiro, Mafalda; Rennig, Maja

    2016-01-01

    flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard “SEVA linkers” for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson...

  7. Construction of Multivalent Homo- and Heterofunctional ABO Blood Group Glycoconjugates Using a Trifunctional Linker Strategy.

    Science.gov (United States)

    Daskhan, Gour Chand; Tran, Hanh-Thuc Ton; Meloncelli, Peter J; Lowary, Todd L; West, Lori J; Cairo, Christopher W

    2018-02-21

    The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with 4 copies of 1 or 2 separate glycan epitopes, providing 4-8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15-18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive N-hydroxysuccinimide (NHS)-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after the removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing the attachment to the NHS-activated poly(ethylene glycol) (PEG) scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne-azide cycloaddition. Using a known tetravalent PEG scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose and the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, 3'-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target B-cell receptors and CD22 receptors on B cells, supporting the

  8. Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo.

    Science.gov (United States)

    Arutla, Viswanath; Leal, Joseph; Liu, Xiaowei; Sokalingam, Sriram; Raleigh, Michael; Adaralegbe, Adejimi; Liu, Li; Pentel, Paul R; Hecht, Sidney M; Chang, Yung

    2017-05-08

    Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC 50 ) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and

  9. NESmapper: accurate prediction of leucine-rich nuclear export signals using activity-based profiles.

    Directory of Open Access Journals (Sweden)

    Shunichi Kosugi

    2014-09-01

    Full Text Available The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs in the cargo proteins, and modulates nuclear-cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS. Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmapper.

  10. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

    Science.gov (United States)

    Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

    2015-06-17

    High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

  11. A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

    Science.gov (United States)

    Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

    2013-11-06

    Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

  12. The Effects of Anchor Groups on (1) TiO2-Catalyzed Photooxidation and (2) Linker-Assisted Assembly on TiO2

    Science.gov (United States)

    Anderson, Ian Mark

    Quantum dot-sensitized solar cells (QDSSCs) are a popular target for research due to their potential for highly efficient, easily tuned absorption. Typically, light is absorbed by quantum dots attached to a semiconductor substrate, such as TiO2, via bifunctional linker molecules. This research aims to create a patterned monolayer of linker molecules on a TiO2 film, which would in turn allow the attachment of a patterned layer of quantum dots. One method for the creation of a patterned monolayer is the functionalization of a TiO2 film with a linker molecule, followed by illumination with a laser at 355 nm. This initiates a TiO 2-catalyzed oxidation reaction, causing loss of surface coverage. A second linker molecule can then be adsorbed onto the TiO2 surface in the illuminated area. Towards that end, the behaviors of carboxylic and phosphonic acids adsorbed on TiO2 have been studied. TiO2 films were functionalized by immersion in solutions a single adsorbate and surface coverage was determined by IR spectroscopy. It is shown that phosphonic acids attain higher surface coverage than carboxylic acids, and will displace them from TiO2 when in a polar solvent. Alkyl chain lengths, which can influence stabilities of monolayers, are shown not to have an effect on this relationship. Equilibrium binding data for the adsorption of n-hexadecanoic acid to TiO2 from a THF solution are presented. It is shown that solvent polarity can affect monolayer stability; carboxylates and phosphonates undergo more desorption into polar solvents than nonpolar. Through illumination, it was possible to remove nearly all adsorbed linkers from TiO2. However, the illuminated areas were found not to be receptive to attachment by a second adsorbate. A possible reason for this behavior is presented. I also report on the synthesis and characterization of a straight-chain, thiol-terminated phosphonic acid. Initial experiments involving monolayer formation and quantum dot attachment are presented

  13. Inverse Effects on Gating and Modulation Caused by a Mutation in the M2-M3 Linker of the GABAA Receptor γ SubunitS⃞

    OpenAIRE

    O'Shea, Sean M.; Williams, Carrie A.; Jenkins, Andrew

    2009-01-01

    M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of α and β subunits of type “A” GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the γ subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engi...

  14. Novel cross-linkers for PDMS networks for controlled and well distributed grafting of functionalities by click chemistry

    DEFF Research Database (Denmark)

    Bahrt, Frederikke; Dimitrov, Ivaylo; Daugaard, Anders Egede

    2013-01-01

    by 35%. The contact angle of PDMS films was increased from 108° to 116° by the introduction of a small poly(pentafluorostyrene) chain. Finally, 17α-ethynyl-1,3,5(10)-estratriene-3,17β-diol and 1-ethynyl-3,5- bis(trifluoromethyl)benzene were incorporated as examples of other functional groups. © 2013......-linkers have been utilized to prepare novel polydimethylsiloxane (PDMS) networks. All functional cross-linkers were successfully incorporated into the networks and were demonstrated to be well distributed within the PDMS films. This was substantiated by fluorescence microscopy of a film prepared with the 4...

  15. Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

    Science.gov (United States)

    Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

    2012-01-01

    Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

  16. Structural models of the different trimers present in the core of phycobilisomes from Gracilaria chilensis based on crystal structures and sequences.

    Directory of Open Access Journals (Sweden)

    Jorge Dagnino-Leone

    Full Text Available Phycobilisomes (PBS are accessory light harvesting protein complexes that directionally transfer energy towards photosystems. Phycobilisomes are organized in a central core and rods radiating from it. Components of phycobilisomes in Gracilaria chilensis (Gch are Phycobiliproteins (PBPs, Phycoerythrin (PE, and Phycocyanin (PC in the rods, while Allophycocyanin (APC is found in the core, and linker proteins (L. The function of such complexes depends on the structure of each component and their interaction. The core of PBS from cyanobacteria is mainly composed by cylinders of trimers of α and β subunits forming heterodimers of Allophycocyanin, and other components of the core including subunits αII and β18. As for the linkers, Linker core (LC and Linker core membrane (LCM are essential for the final emission towards photoreaction centers. Since we have previously focused our studies on the rods of the PBS, in the present article we investigated the components of the core in the phycobilisome from the eukaryotic algae, Gracilaria chilensis and their organization into trimers. Transmission electron microscopy provided the information for a three cylinders core, while the three dimensional structure of Allophycocyanin purified from Gch was determined by X-ray diffraction method and the biological unit was determined as a trimer by size exclusion chromatography. The protein sequences of all the components of the core were obtained by sequencing the corresponding genes and their expression confirmed by transcriptomic analysis. These subunits have seldom been reported in red algae, but not in Gracilaria chilensis. The subunits not present in the crystallographic structure were modeled to build the different composition of trimers. This article proposes structural models for the different types of trimers present in the core of phycobilisomes of Gch as a first step towards the final model for energy transfer in this system.

  17. Draft genome sequence of Lampropedia cohaerens strain CT6(T) isolated from arsenic rich microbial mats of a Himalayan hot water spring.

    Science.gov (United States)

    Tripathi, Charu; Mahato, Nitish K; Rani, Pooja; Singh, Yogendra; Kamra, Komal; Lal, Rup

    2016-01-01

    Lampropedia cohaerens strain CT6(T), a non-motile, aerobic and coccoid strain was isolated from arsenic rich microbial mats (temperature ~45 °C) of a hot water spring located atop the Himalayan ranges at Manikaran, India. The present study reports the first genome sequence of type strain CT6(T) of genus Lampropedia cohaerens. Sequencing data was generated using the Illumina HiSeq 2000 platform and assembled with ABySS v 1.3.5. The 3,158,922 bp genome was assembled into 41 contigs with a mean GC content of 63.5 % and 2823 coding sequences. Strain CT6(T) was found to harbour genes involved in both the Entner-Duodoroff pathway and non-phosphorylated ED pathway. Strain CT6(T) also contained genes responsible for imparting resistance to arsenic, copper, cobalt, zinc, cadmium and magnesium, providing survival advantages at a thermal location. Additionally, the presence of genes associated with biofilm formation, pyrroloquinoline-quinone production, isoquinoline degradation and mineral phosphate solubilisation in the genome demonstrate the diverse genetic potential for survival at stressed niches.

  18. Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

    Directory of Open Access Journals (Sweden)

    Blackmon Barbara P

    2011-07-01

    Full Text Available Abstract Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

  19. Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

    Science.gov (United States)

    Costa, M G S; Silva, Y F; Batista, P R

    2018-03-14

    Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

  20. Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

    Science.gov (United States)

    Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João M; Belizário, José E

    2017-12-01

    Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD 198 ↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (k cat /K M ) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

  1. Synthesis of highly fluorescent and thio-linkers stabilize gold quantum dots and nano clusters in DMF for bio-labeling

    Energy Technology Data Exchange (ETDEWEB)

    Rastogi, Shiva K., E-mail: srastogi@uidaho.edu [University of Idaho, Department of Chemistry (United States); Denn, Benjamin D.; Branen, A. Larry [University of Idaho, Coeur D' Alene, Biosensors and Nanotechnology Application Laboratory (BNAL) (United States)

    2012-01-15

    This study demonstrates a one versus two-step synthesis of fluorescent gold quantum dots (F-AuQDs) and nano clusters (F-AuNCs) functionalized with thiolated organic linkers using reduction of gold precursor in N,N Prime -dimethylformamide in 1 h of reaction. The F-AuQDs and F-AuNCs show fluorescence emission at 425 {+-} 5 nm upon excitation at 345 {+-} 5 nm of wavelength, with good water solubility and stability. Five different thiolated organic binary linkers consisting of various functional groups including: carboxylic acid, hydroxyl, and aromatic amine, were conjugated with the F-AuQDs and F-AuNCs. The formation mechanism and functionalization of the F-AuQDs and F-AuNCs was characterized using UV-vis absorption spectra, UV-vis light, fluorescent emission spectra, pH, TEM, and FTIR. The fluorescence emission of the F-AuQDs and F-AuNCs is greatly dependent on the thio-linker. This novel one-step approach provides facile and fast synthesis of F-AuQDs and F-AuNCs over the two-step method, with less than 5 h of reaction and workup compared to more than 28 h of reaction for the two-step approach. These thio-linker functionalized F-AuQDs and F-AuNCs have a wide application in fluorescent labeling of biomolecules, optical devices, imaging, energy transfer, and biosensing.

  2. Serum Stabilities of Short Tryptophan-and Arginine-Rich Antimicrobial Peptide Analogs

    NARCIS (Netherlands)

    Nguyen, L.T.; Chau, J.K.; Perry, N.A.; de Boer, L.; Zaat, S.A.J.; Vogel, H.J.

    2010-01-01

    Background: Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the "antimicrobial

  3. Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki

    2014-01-01

    , which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide Gal...

  4. Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

    Science.gov (United States)

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2014-11-13

    The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

  5. Effects of linker variation on the in vitro and in vivo characteristics of an 111In-labeled RGD peptide

    International Nuclear Information System (INIS)

    Dijkgraaf, Ingrid; Liu, Shuang; Kruijtzer, John A.W.; Soede, Annemieke C.; Oyen, Wim J.G.; Liskamp, Rob M.J.; Corstens, Frans H.M.; Boerman, Otto C.

    2007-01-01

    Introduction: Due to the selective expression of the α v β 3 integrin in tumors, radiolabeled arginine-glycine-aspartic acid (RGD) peptides are attractive candidates for tumor targeting. Minor modifications of these peptides could have a major impact on in vivo characteristics. In this study, we systematically investigated the effects of linker modification between two cyclic RGD sequences and DOTA (1,4,7,10-tetraazadodecane-N,N',N ' ,N'''-tetraacetic acid) on the in vitro and in vivo characteristics of the tracer. Methods: A dimeric RGD peptide was synthesized and conjugated either directly with DOTA or via different linkers: PEG 4 (polyethylene glycol), glutamic acid or lysine. The RGD peptides were radiolabeled with 111 In, and their in vitro and in vivo α v β 3 -binding characteristics were determined. Results: LogP values varied between -2.82±0.06 and -3.95±0.33. The IC 50 values for DOTA-E-[c(RGDfK)] 2 , DOTA-PEG 4 -E-[c(RGDfK)] 2 , DOTA-E-E-[c(RGDfK)] 2 and DOTA-K-E-[c(RGDfK)] 2 were comparable. Two hours after injection, the tumor uptakes of the 111 In-labeled compounds were not significantly different. The kidney accumulation of [ 111 In]-DOTA-K-E-[c(RGDfK)] 2 [4.05±0.20% of the injected dose per gram (ID/g)] was significantly higher as compared with that of [ 111 In]-DOTA-E-[c(RGDfK)] 2 (2.63±0.19% ID/g; P 111 In]-DOTA-E-E-[c(RGDfK)] 2 (2.16±0.21% ID/g; P 111 In]-DOTA-E-E-[c(RGDfK)] 2 (2.12±0.09% ID/g) was significantly higher as compared with that of [ 111 In]-DOTA-E-[c(RGDfK)] 2 (1.64±0.1% ID/g; P 111 In]-DOTA-K-E-[c(RGDfK)] 2 (1.52±0.04% ID/g; P v β 3 and tumor uptake. Insertion of lysine caused enhanced kidney retention; that of glutamic acid also resulted in enhanced retention in the kidneys. PEG 4 appeared to be the most suitable linker as compared with glutamic acid and lysine because it has the highest tumor-to-blood ratio and the lowest uptake in the kidney and liver

  6. Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima.

    Science.gov (United States)

    Ganal, M; Hemleben, V

    1986-11-01

    The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349-352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168-169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.

  7. Preparation and in vivo evaluation of linkers for 211At labeling of humanized anti-Tac

    International Nuclear Information System (INIS)

    Yordanov, A.T.; Garmestani, K.; Zhang, M.; Zhang, Z.; Yao, Z.; Phillips, K.E.; Herring, B.; Horak, E.; Beitzel, M.P.; Schwarz, U.P.; Gansow, O.A.; Plascjak, P.S.; Eckelman, W.C.; Waldmann, T.A.; Brechbiel, M.W.

    2001-01-01

    The syntheses, radiolabeling, antibody conjugation, and in vivo evaluation of new linkers for 211 At labeling of humanized anti-Tac (Hu-anti-Tac), an antibody to the α-chain of the IL-2 receptor (IL-2Rα) shown to be a useful target for radioimmunotherapy are described. Synthesis of the organometallic linker precursors is accomplished by reaction of the corresponding bromo- or iodoaryl esters with bis(tributyltin) in the presence of a palladium catalyst. Subsequent conversion to the corresponding N-succinimidyl ester and labeling with 211 At of two new linkers, N-succinimidyl 4-[ 211 At]astato-3-methylbenzoate and N-succinimidyl N-(4-[ 211 At]astatophenethyl)succinamate (SAPS), together with the previously reported N-succinimidyl 4-[ 211 At]astatobenzoate and N-succinimidyl 3-[ 211 At]astato-4-methylbenzoate, are each conjugated to Hu-anti-Tac. The plasma survival times of these conjugates are compared to those of directly iodinated ( 125 I) Hu-anti-Tac. The N-succinimidyl N-(4-[ 211 At]astatophenethyl)succinamate compound (SAPS) emerged from this assay as the most viable candidate for 211 At-labeling of Hu-anti-Tac. SAPS, along with the directly analogous radio-iodinated reagent, N-succinimidyl N-(4-[ 125 I]astatophenethyl)succinamate (SIPS), are evaluated in a biodistribution study along with directly iodinated ( 125 I) Hu-anti-Tac. Blood clearance and biological accretion results indicate that SAPS is a viable candidate for further evaluation for radioimmunotherapy of cancer

  8. Doxorubicin conjugation and drug linker chemistry alter the intravenous and pulmonary pharmacokinetics of a PEGylated Generation 4 polylysine dendrimer in rats.

    Science.gov (United States)

    Leong, Nathania J; Mehta, Dharmini; McLeod, Victoria M; Kelly, Brian D; Pathak, Rashmi; Owen, David J; Porter, Christopher Jh; Kaminskas, Lisa M

    2018-05-28

    PEGylated polylysine dendrimers have demonstrated potential as inhalable drug delivery systems that can improve the treatment of lung cancers. Their treatment potential may be enhanced by developing constructs that display prolonged lung retention, together with good systemic absorption, the capacity to passively target lung tumours from the blood and highly selective, yet rapid liberation in the tumour microenvironment. This study sought to characterise how the nature of cathepsin B cleavable peptide linkers, used to conjugate doxorubicin to a PEGylated (PEG570) G4 polylysine dendrimer, affect drug liberation kinetics and intravenous and pulmonary pharmacokinetics in rats. The construct bearing a self-emolative diglycolic acid-V-Citrulline linker exhibited faster doxorubicin release kinetics compared to constructs bearing self emolative diglycolic acid-GLFG, or non-self emolative glutaric acid-GLFG linkers. The V-Citrulline construct exhibited slower plasma clearance, but faster absorption from the lungs than a GLFG construct, although mucociliary clearance and urinary elimination were unchanged. Doxorubicin-conjugation enhanced localisation in the bronchoalveolar lavage fluid compared to lung tissue, suggesting that projection of doxorubicin from the dendrimer surface reduced tissue uptake. These data show that the linker chemistry employed to conjugate drugs to PEGylated carriers can affect drug release profiles and systemic and lung disposition. Copyright © 2018. Published by Elsevier Inc.

  9. Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli.

    Science.gov (United States)

    Buchner, Sophie; Schlundt, Andreas; Lassak, Jürgen; Sattler, Michael; Jung, Kirsten

    2015-07-31

    The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and α-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pHpH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The environmental impacts on the star formation main sequence: An Hα study of the newly discovered rich cluster at z = 1.52

    Energy Technology Data Exchange (ETDEWEB)

    Koyama, Yusei; Kodama, Tadayuki; Tadaki, Ken-ichi; Hayashi, Masao [National Astronomical Observatory of Japan, Mitaka, Tokyo 181-8588 (Japan); Tanaka, Ichi [Subaru Telescope, National Astronomical Observatory of Japan, 650 North A' ohoku Place, Hilo, HI 96720 (United States); Shimakawa, Rhythm, E-mail: koyama.yusei@nao.ac.jp [Department of Astronomical Science, The Graduate University for Advanced Studies, Mitaka, Tokyo 181-8588 (Japan)

    2014-07-01

    We report the discovery of a strong over-density of galaxies in the field of a radio galaxy at z = 1.52 (4C 65.22) based on our broadband and narrow-band (Hα) photometry with the Subaru Telescope. We find that Hα emitters are located in the outskirts of the density peak (cluster core) dominated by passive red-sequence galaxies. This resembles the situation in lower-redshift clusters, suggesting that the newly discovered structure is a well-evolved rich galaxy cluster at z = 1.5. Our data suggest that the color-density and stellar mass-density relations are already in place at z ∼ 1.5, mostly driven by the passive red massive galaxies residing within r{sub c} ≲ 200 kpc from the cluster core. These environmental trends almost disappear when we consider only star-forming (SF) galaxies. We do not find SFR-density or SSFR-density relations amongst SF galaxies, and the location of the SF main sequence does not significantly change with environment. Nevertheless, we find a tentative hint that star-bursting galaxies (up-scattered objects from the main sequence) are preferentially located in a small group at ∼1 Mpc away from the main body of the cluster. We also argue that the scatter of the SF main sequence could be dependent on the distance to the nearest neighboring galaxy.

  11. Mixed-linker zeolitic imidazolate framework mixed-matrix membranes for aggressive CO2 separation from natural gas

    KAUST Repository

    Thompson, Joshua A.; Vaughn, Justin T.; Brunelli, Nicholas A.; Koros, William J.; Jones, Christopher W.; Nair, Sankar

    2014-01-01

    Zeolitic imidazolate framework (ZIF) materials are a promising subclass of metal-organic frameworks (MOF) for gas separations. However, due to the deleterious effects of gate-opening phenomena associated with organic linker rotation near

  12. GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Liachko, Ivan; Youngblood, Rachel A; Tsui, Kyle; Bubb, Kerry L; Queitsch, Christine; Raghuraman, M K; Nislow, Corey; Brewer, Bonita J; Dunham, Maitreya J

    2014-03-01

    The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.

  13. Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z.

    Directory of Open Access Journals (Sweden)

    Makenzie Saoura

    Full Text Available The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332 at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.

  14. Agaricus bisporus genome sequence: a commentary.

    Science.gov (United States)

    Kerrigan, Richard W; Challen, Michael P; Burton, Kerry S

    2013-06-01

    The genomes of two isolates of Agaricus bisporus have been sequenced recently. This soil-inhabiting fungus has a wide geographical distribution in nature and it is also cultivated in an industrialized indoor process ($4.7bn annual worldwide value) to produce edible mushrooms. Previously this lignocellulosic fungus has resisted precise econutritional classification, i.e. into white- or brown-rot decomposers. The generation of the genome sequence and transcriptomic analyses has revealed a new classification, 'humicolous', for species adapted to grow in humic-rich, partially decomposed leaf material. The Agaricus biporus genomes contain a collection of polysaccharide and lignin-degrading genes and more interestingly an expanded number of genes (relative to other lignocellulosic fungi) that enhance degradation of lignin derivatives, i.e. heme-thiolate peroxidases and β-etherases. A motif that is hypothesized to be a promoter element in the humicolous adaptation suite is present in a large number of genes specifically up-regulated when the mycelium is grown on humic-rich substrate. The genome sequence of A. bisporus offers a platform to explore fungal biology in carbon-rich soil environments and terrestrial cycling of carbon, nitrogen, phosphorus and potassium. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Genome Sequence of a Novel Archaeal Rudivirus Recovered from a Mexican Hot Spring

    DEFF Research Database (Denmark)

    Servín-Garcidueñas, L; Peng, X; Garrett, R

    2013-01-01

    We report the consensus genome sequence of a novel GC-rich rudivirus, designated SMR1 (Sulfolobales Mexican rudivirus 1), assembled from a high-throughput sequenced environmental sample from a hot spring in Los Azufres National Park in western Mexico.......We report the consensus genome sequence of a novel GC-rich rudivirus, designated SMR1 (Sulfolobales Mexican rudivirus 1), assembled from a high-throughput sequenced environmental sample from a hot spring in Los Azufres National Park in western Mexico....

  16. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers

    Directory of Open Access Journals (Sweden)

    Quail Michael A

    2012-07-01

    Full Text Available Abstract Background Next generation sequencing (NGS technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Results Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. Conclusions All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

  17. GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Ivan Liachko

    2014-03-01

    Full Text Available The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.

  18. Characterization and sequence analysis of cysteine and glycine-rich ...

    African Journals Online (AJOL)

    Tarek

    2011-04-18

    Apr 18, 2011 ... nucleotide alignment of both native buffalo and cattle CSRP3 cDNAs sequences ..... Exon III, Identities = 71/75 (94%), Gaps = 1/75 (1%) Strand=Plus/Plus ... Band MR, Larson JH, Rebeiz M, Green CA, Heyen DW, Donovan J,.

  19. The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

    Science.gov (United States)

    Iacobucci, Claudio; Piotrowski, Christine; Rehkamp, Anne; Ihling, Christian H.; Sinz, Andrea

    2018-04-01

    Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. [Figure not available: see fulltext.

  20. Ab initio study of hydrogen adsorption on benzenoid linkers in metal-organic framework materials

    International Nuclear Information System (INIS)

    Gao Yi; Zeng, X C

    2007-01-01

    We have computed the energies of adsorption of molecular hydrogen on a number of molecular linkers in metal-organic framework solid materials using density functional theory (DFT) and ab initio molecular orbital methods. We find that the hybrid B3LYP (Becke three-parameter Lee-Yang-Parr) DFT method gives a qualitatively incorrect prediction of the hydrogen binding with benzenoid molecular linkers. Both local-density approximation (LDA) and generalized gradient approximation (GGA) DFT methods are inaccurate in predicting the values of hydrogen binding energies, but can give a qualitatively correct prediction of the hydrogen binding. When compared to the more accurate binding-energy results based on the ab initio Moeller-Plesset second-order perturbation (MP2) method, the LDA results may be viewed as an upper limit while the GGA results may be viewed as a lower limit. Since the MP2 calculation is impractical for realistic metal-organic framework systems, the combined LDA and GGA calculations provide a cost-effective way to assess the hydrogen binding capability of these systems

  1. Properties and distribution of pure GA-sequences of mammalian genomes.

    Directory of Open Access Journals (Sweden)

    Guenter Albrecht-Buehler

    Full Text Available The article describes DNA sequences of mammalian genomes that are longer than 50 bases, but consist exclusively of G's and A's ('pure GA-sequences'. Although their frequency of incidence should be 10(-16 or smaller, the chromosomes of human, chimpanzee, dog, cat, rat, and mouse contained many tens of thousands of them ubiquitously located along the chromosomes with a species-dependent density, reaching sizes of up to 1300 [b]. With the exception of a small number of poly-A-, poly-G-, poly-GA-, and poly-GAAA-sequences (combined <0.5%, all pure GA-sequences of the mammals tested were unique individuals, contained several repeated short GA-containing motifs, and shared a common hexa-nucleotide spectrum. At most 2% of the human GA-sequences were transcribed into mRNAs; all others were not coding for proteins. Although this could have made them less subject to natural selection, they contained many [corrected] times fewer point mutations than one should expect from the genome at large. As to the presence of other sequences with similarly restricted base contents, there were approximately as many pure TC-sequences as pure GA-sequences, but many fewer pure AC-, TA, and TG-sequences. There were practically no pure GC-sequences. The functions of pure GA-sequences are not known. Supported by a number of observations related to heat shock phenomena, the article speculates that they serve as genomic sign posts which may help guide polymerases and transcription factors to their proper targets, and/or as spatial linkers that help generate the 3-dimensional organization of chromatin.

  2. Unusual and highly tunable missing-linker defects in zirconium metal-organic framework UiO-66 and their important effects on gas adsorption.

    Science.gov (United States)

    Wu, Hui; Chua, Yong Shen; Krungleviciute, Vaiva; Tyagi, Madhusudan; Chen, Ping; Yildirim, Taner; Zhou, Wei

    2013-07-17

    UiO-66 is a highly important prototypical zirconium metal-organic framework (MOF) compound because of its excellent stabilities not typically found in common porous MOFs. In its perfect crystal structure, each Zr metal center is fully coordinated by 12 organic linkers to form a highly connected framework. Using high-resolution neutron power diffraction technique, we found the first direct structural evidence showing that real UiO-66 material contains significant amount of missing-linker defects, an unusual phenomenon for MOFs. The concentration of the missing-linker defects is surprisingly high, ∼10% in our sample, effectively reducing the framework connection from 12 to ∼11. We show that by varying the concentration of the acetic acid modulator and the synthesis time, the linker vacancies can be tuned systematically, leading to dramatically enhanced porosity. We obtained samples with pore volumes ranging from 0.44 to 1.0 cm(3)/g and Brunauer-Emmett-Teller surface areas ranging from 1000 to 1600 m(2)/g, the largest values of which are ∼150% and ∼60% higher than the theoretical values of defect-free UiO-66 crystal, respectively. The linker vacancies also have profound effects on the gas adsorption behaviors of UiO-66, in particular CO2. Finally, comparing the gas adsorption of hydroxylated and dehydroxylated UiO-66, we found that the former performs systematically better than the latter (particularly for CO2) suggesting the beneficial effect of the -OH groups. This finding is of great importance because hydroxylated UiO-66 is the practically more relevant, non-air-sensitive form of this MOF. The preferred gas adsorption on the metal center was confirmed by neutron diffraction measurements, and the gas binding strength enhancement by the -OH group was further supported by our first-principles calculations.

  3. Micromachined silicon acoustic delay line with 3D-printed micro linkers and tapered input for improved structural stability and acoustic directivity

    International Nuclear Information System (INIS)

    Cho, Y; Kumar, A; Xu, S; Zou, J

    2016-01-01

    Recent studies have shown that micromachined silicon acoustic delay lines can provide a promising solution to achieve real-time photoacoustic tomography without the need for complex transducer arrays and data acquisition electronics. To achieve deeper imaging depth and wider field of view, a longer delay time and therefore delay length are required. However, as the length of the delay line increases, it becomes more vulnerable to structural instability due to reduced mechanical stiffness. In this paper, we report the design, fabrication, and testing of a new silicon acoustic delay line enhanced with 3D printed polymer micro linker structures. First, mechanical deformation of the silicon acoustic delay line (with and without linker structures) under gravity was simulated by using finite element method. Second, the acoustic crosstalk and acoustic attenuation caused by the polymer micro linker structures were evaluated with both numerical simulation and ultrasound transmission testing. The result shows that the use of the polymer micro linker structures significantly improves the structural stability of the silicon acoustic delay lines without creating additional acoustic attenuation and crosstalk. In addition, the improvement of the acoustic acceptance angle of the silicon acoustic delay lines was also investigated to better suppress the reception of unwanted ultrasound signals outside of the imaging plane. These two improvements are expected to provide an effective solution to eliminate current limitations on the achievable acoustic delay time and out-of-plane imaging resolution of micromachined silicon acoustic delay line arrays. (paper)

  4. Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

    DEFF Research Database (Denmark)

    Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld

    1992-01-01

    A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...... areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155...... between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses. The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA....

  5. IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

    Science.gov (United States)

    Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

    2017-06-26

    IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

  6. Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

    Science.gov (United States)

    Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

    2015-02-25

    In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. PCR Assays for Identification of Coccidioides posadasii Based on the Nucleotide Sequence of the Antigen 2/Proline-Rich Antigen

    Science.gov (United States)

    Bialek, Ralf; Kern, Jan; Herrmann, Tanja; Tijerina, Rolando; Ceceñas, Luis; Reischl, Udo; González, Gloria M.

    2004-01-01

    A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens. PMID:14766853

  8. Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

    Science.gov (United States)

    Rackwitz, Jenny; Bald, Ilko

    2018-03-26

    During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Identification of a minimal functional linker in human topoisomerase I by domain swapping with Cre recombinase

    DEFF Research Database (Denmark)

    Hougaard, Rikke Frøhlich; Juul, Sissel; Vinther, Maria

    2008-01-01

    . In this study we replace 86 amino acids including the linker domain of the cellular type IB topoisomerase, human topoisomerase I, with four, six, or eight amino acids from the corresponding short loop region in Cre recombinase. In vitro characterization of the resulting chimeras, denoted Cropos, reveals...

  10. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    International Nuclear Information System (INIS)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

    1990-01-01

    A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

  11. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  12. Glassin, a histidine-rich protein from the siliceous skeletal system of the marine sponge Euplectella, directs silica polycondensation.

    Science.gov (United States)

    Shimizu, Katsuhiko; Amano, Taro; Bari, Md Rezaul; Weaver, James C; Arima, Jiro; Mori, Nobuhiro

    2015-09-15

    The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.

  13. Radioiodination of protein using 2,3,5,6-tetrafluorophenyl 3-(nido-carboranyl) propionate (TCP) as a potential bi-functional linker: Synthesis and biodistribution in mice

    International Nuclear Information System (INIS)

    Lin Rushan; Liu Ning; Yang Yuanyou; Li Bing; Liao Jiali; Jin Jiannan

    2009-01-01

    2,3,5,6-Tetrafluorophenyl 3-(nido-carboranyl) propionate (TCP), as a new potential bi-functional linker for radiohalogenation of proteins or peptides, was synthesized. With this bi-functional linker, the first attempt to conjugate bovine serum albumin (BSA) with 125 I was made and the biodistribution of the conjugated BSA ( 125 I-TCP-BSA) was investigated in NIH strain mice. By the use of TCP as the linker, BSA was conjugated with 125 I in a labeling yield of 58-75% and with radiochemical purity of 99.8% after purification by Sephadex TM G-50. Even after being kept at room temperature for 72 h, the radiochemical purity of 125 I-TCP-BSA was still more than 98%, much higher than that of the directly 125 I-labeled BSA ( 125 I-BSA). Meanwhile, biodistribution experiments in mice indicated that the uptake of 125 I with 125 I-TCP-BSA into thyroid was obviously less than that with 125 I-BSA post-injection. All the results implied that the 125 I-conjugated BSA ( 125 I-TCP-BSA) was considerably stable in vivo as well as in vitro, and TCP was regarded as a promising bi-functional linker for radiohalogenation of proteins

  14. Geologic map showing springs rich in carbon dioxide or or chloride in California

    Science.gov (United States)

    Barnes, Ivan; Irwin, William P.; Gibson, H.A.

    1975-01-01

    Carbon dioxide- and chloride-rich springs occur in all geologic provinces in California, but are most abundant in the Coast Ranges and the Great Valley. The carbon-dioxide-rich springs issue mainly from Franciscan terrane; they also are rich in boron and are of the metamorphic type (White, 1957). Based on isotopic data, either the carbon dioxide or the water, or both, may be of metamorphic origin. Because of high magnesium values, the water of many of the carbon-dioxide-rich springs is thought to have passed through serpentinite. The chloride-rich waters are most common in rocks of the Great Valley sequence. Nearly all are more dilute than present-day sea water. The similarity in isotopic compositions of the metamorphic carbon-dioxide-rich water and the chloride-rich water may indicate a similar extent of water-rock interaction.

  15. Hybrid Zeolitic Imidazolate Frameworks: Controlling Framework Porosity and Functionality by Mixed-Linker Synthesis

    KAUST Repository

    Thompson, Joshua A.

    2012-05-22

    Zeolitic imidazolate frameworks (ZIFs) are a subclass of nanoporous metal-organic frameworks (MOFs) that exhibit zeolite-like structural topologies and have interesting molecular recognition properties, such as molecular sieving and gate-opening effects associated with their pore apertures. The synthesis and characterization of hybrid ZIFs with mixed linkers in the framework are described in this work, producing materials with properties distinctly different from the parent frameworks (ZIF-8, ZIF-90, and ZIF-7). NMR spectroscopy is used to assess the relative amounts of the different linkers included in the frameworks, whereas nitrogen physisorption shows the evolution of the effective pore size distribution in materials resulting from the framework hybridization. X-ray diffraction shows these hybrid materials to be crystalline. In the case of ZIF-8-90 hybrids, the cubic space group of the parent frameworks is continuously maintained, whereas in the case of the ZIF-7-8 hybrids there is a transition from a cubic to a rhombohedral space group. Nitrogen physisorption data reveal that the hybrid materials exhibit substantial changes in gate-opening phenomena, either occurring at continuously tunable partial pressures of nitrogen (ZIF-8-90 hybrids) or loss of gate-opening effects to yield more rigid frameworks (ZIF-7-8 hybrids). With this synthetic approach, significant alterations in MOF properties may be realized to suit a desired separation or catalytic process. © 2012 American Chemical Society.

  16. A Switchable Linker-Based Immunoassay for Ultrasensitive Visible Detection of Salmonella in Tomatoes.

    Science.gov (United States)

    Hahn, Jungwoo; Kim, Eunghee; You, Young Sang; Gunasekaran, Sundaram; Lim, Seokwon; Choi, Young Jin

    2017-10-01

    On-site detection for sensitive identification of foodborne pathogens on fresh produce with minimal use of specialized instrumentation is crucial to the food industry. A switchable linker (SL)-based immunoassay was designed for ultrasensitive on-site detection of Salmonella in tomato samples. The assay is based on large-scale aggregation of gold nanoparticles (GNPs), induced by a quantitative relationship among the biotinylated Salmonella polyclonal antibody (b-Ab) used as the SL, the functionalized GNPs, and Salmonella. Important factors such as the concentration of SLs, time required for large-scale aggregation, and selectivity of b-Ab were optimized to minimize the detection time (within 45 min with gentle agitation) and achieve the lowest limit of detection (LOD; 10 CFU/g in tomato samples) possible. This SL-based immunoassay with its relatively low LOD and short detection time may meet the need for rapid, simple, on-site analysis of pathogens in fresh produce. The novel switchable linker-based immunoassay is a rapid, specific, and sensitive method that has potential applications for routine diagnostics of Salmonella in tomato products. These advantages make it a practical approach for general use in the processing industry to detect Salmonella rapidly and to implement appropriate regulatory procedures. Furthermore, it could be applied to other fresh products including cantaloupe, strawberry, and cucumbers. © 2017 Institute of Food Technologists®.

  17. The decorin sequence SYIRIADTNIT binds collagen type I

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Oldberg, Ake

    2007-01-01

    Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site......-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro....... These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins....

  18. Giardia telomeric sequence d(TAGGG)4 forms two intramolecular G-quadruplexes in K+ solution: effect of loop length and sequence on the folding topology.

    Science.gov (United States)

    Hu, Lanying; Lim, Kah Wai; Bouaziz, Serge; Phan, Anh Tuân

    2009-11-25

    Recently, it has been shown that in K(+) solution the human telomeric sequence d[TAGGG(TTAGGG)(3)] forms a (3 + 1) intramolecular G-quadruplex, while the Bombyx mori telomeric sequence d[TAGG(TTAGG)(3)], which differs from the human counterpart only by one G deletion in each repeat, forms a chair-type intramolecular G-quadruplex, indicating an effect of G-tract length on the folding topology of G-quadruplexes. To explore the effect of loop length and sequence on the folding topology of G-quadruplexes, here we examine the structure of the four-repeat Giardia telomeric sequence d[TAGGG(TAGGG)(3)], which differs from the human counterpart only by one T deletion within the non-G linker in each repeat. We show by NMR that this sequence forms two different intramolecular G-quadruplexes in K(+) solution. The first one is a novel basket-type antiparallel-stranded G-quadruplex containing two G-tetrads, a G x (A-G) triad, and two A x T base pairs; the three loops are consecutively edgewise-diagonal-edgewise. The second one is a propeller-type parallel-stranded G-quadruplex involving three G-tetrads; the three loops are all double-chain-reversal. Recurrence of several structural elements in the observed structures suggests a "cut and paste" principle for the design and prediction of G-quadruplex topologies, for which different elements could be extracted from one G-quadruplex and inserted into another.

  19. An AU-rich element in the 3{prime} untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qiuyun; Adams, C.C.; Usack, L. [Cornell Univ., Ithaca, NY (United States)] [and others

    1995-04-01

    In chloroplasts, the 3{prime} untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3{prime}-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3{prime} IR-containing RNA (3{prime} IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b{sub 6}/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T{sub 1} digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3{prime} IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3{prime} untranslated region, only a single copy, which we have termed box II, appears to be essential for in vivo protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3{prime}-end processing and/or influence the stability of petD mRNA in chloroplasts. 48 refs., 9 figs., 2 tabs.

  20. Simple, Fast and Selective Detection of Adenosine Triphosphate at Physiological pH Using Unmodified Gold Nanoparticles as Colorimetric Probes and Metal Ions as Cross-Linkers

    Directory of Open Access Journals (Sweden)

    Huan Pang

    2012-11-01

    Full Text Available We report a simple, fast and selective colorimetric assay of adenosine triphosphate (ATP using unmodified gold nanoparticles (AuNPs as probes and metal ions as cross-linkers. ATP can be assembled onto the surface of AuNPs through interaction between the electron-rich nitrogen atoms and the electron-deficient surface of AuNPs. Accordingly, Cu2+ ions induce a change in the color and UV/Vis absorbance of AuNPs by coordinating to the triphosphate groups and a ring nitrogen of ATP. A detection limit of 50 nM was achieved, which is comparable to or lower than that achievable by the currently used electrochemical, spectroscopic or chromatographic methods. The theoretical simplicity and high selectivity reported herein demonstrated that AuNPs-based colorimetric assay could be applied in a wide variety of fields by rationally designing the surface chemistry of AuNPs. In addition, our results indicate that ATP-modified AuNPs are less stable in Cu2+, Cd2+ or Zn2+-containing solutions due to the formation of the corresponding dimeric metal-ATP complexes.

  1. Dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like peptide-1 receptor ligands.

    Science.gov (United States)

    Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M

    2015-02-18

    Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents.

  2. Next generation sequencing reveals the hidden diversity of zooplankton assemblages.

    Directory of Open Access Journals (Sweden)

    Penelope K Lindeque

    Full Text Available BACKGROUND: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel. METHODOLOGY/PRINCIPLE FINDINGS: Plankton net hauls (200 µm were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups. CONCLUSIONS: Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may

  3. A magnetic nanoparticle-clustering biosensor for blu-ray based optical detection of small-molecules

    DEFF Research Database (Denmark)

    Yang, Jaeyoung; Donolato, Marco; Antunes, Paula Soares Martins

    2014-01-01

    MNP-clustering facilitates high-resolution small-molecule assays. For experiments, aptamer-functionalized MNPs (Apt-MNPs) were first incubated with adenosine-5'-triphosphate (ATP) followed by adding MNPs with linker strands (linker-MNPs). The linker hybridizes with a region of aptamer sequences...

  4. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  5. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    Directory of Open Access Journals (Sweden)

    Shih Ping Yao

    2002-04-01

    Full Text Available Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C, is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57 of transgenic pigs (F0 generation. Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

  6. Improved i-motif thermal stability by insertion of anthraquinone monomers

    DEFF Research Database (Denmark)

    Gouda, Alaa S; Amine, Mahasen S.; Pedersen, Erik Bjerregaard

    2017-01-01

    In order to gain insight into how to improve thermal stability of i-motifs when used in the context of biomedical and nanotechnological applications, novel anthraquinone-modified i-motifs were synthesized by insertion of 1,8-, 1,4-, 1,5- and 2,6-disubstituted anthraquinone monomers into the TAA...... loops of a 22mer cytosine-rich human telomeric DNA sequence. The influence of the four anthraquinone linkers on the i-motif thermal stability was investigated at 295 nm and pH 5.5. Anthraquinone monomers modulate the i-motif stability in a position-depending manner and the modulation also depends...... unlocked nucleic acid monomers or twisted intercalating nucleic acid. The 2,6-disubstituted anthraquinone linker replacing T10 enabled a significant increase of i-motif thermal melting by 8.2 °C. A substantial increase of 5.0 °C in i-motif thermal melting was recorded when both A6 and T16 were modified...

  7. A Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide-1 Receptor Ligands

    Science.gov (United States)

    Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M.

    2016-01-01

    Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using the glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel alpha helix stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enables this technique to potentially be used as a general method for labeling alpha helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents. PMID:25594741

  8. Mechanistic Evaluation of Motion in Redox-Driven Rotaxanes Reveals Longer Linkers Hasten Forward Escape's and Hinder Backward Translations

    DEFF Research Database (Denmark)

    Andersen, S. S.; Share, A. I.; Poulsen, B. L.

    2014-01-01

    temperatures to provide activation enthalpies (Delta H-double dagger) and entropies (Delta S-double dagger). Longer glycol linkers led to modest increases in the forward escape (t(1/2) = 60 to 69 s); though not because of a diffusive walk. The reduced rate of motion backward depended on folded structures...

  9. Dibenzotetraaza[14]annulene-adenine conjugate recognizes complementary poly dT among ss-DNA/ss-RNA sequences.

    Science.gov (United States)

    Radić Stojković, Marijana; Škugor, Marko; Tomić, Sanja; Grabar, Marina; Smrečki, Vilko; Dudek, Łukasz; Grolik, Jarosław; Eilmes, Julita; Piantanida, Ivo

    2013-06-28

    Among three novel DBTAA derivatives only the DBTAA-propyl-adenine conjugate showed recognition of the consecutive oligo dT sequence by increased affinity and specific induced chirooptical response in comparison to other single stranded RNA and DNA; whereby of particular importance is the up until now unique efficient differentiation between dT and rU. At variance, its close analogue DBTAA-hexyl-adenine did not reveal any selectivity between ss-DNA/RNA pointing out the important role of steric factors (linker length); moreover non-selectivity of the reference compound (, lacking adenine) stressed the importance of adenine interactions in the selectivity.

  10. Butane-1,2,3,4-tetraol-based amphiphilic stereoisomers for membrane protein study: importance of chirality in the linker region

    DEFF Research Database (Denmark)

    Das, Manabendra; Du, Yang; Mortensen, Jonas S.

    2017-01-01

    of the targeted membrane proteins depending on the chirality of the linker region. These findings indicate an important role for detergent stereochemistry in membrane protein stabilization. In addition, we generally observed enhanced detergent efficacy with increasing alkyl chain length, reinforcing...

  11. Classification, expression pattern and comparative analysis of sugarcane expressed sequences tags (ESTs encoding glycine-rich proteins (GRPs

    Directory of Open Access Journals (Sweden)

    Fusaro Adriana

    2001-01-01

    Full Text Available Since the isolation of the first glycine-rich proteins (GRPs in plants a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localization of some GRP groups, clearly indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights into the molecular biology and cell biology of plants. Complexly regulated promoters and distinct mechanisms for the regulation of gene expression have been demonstrated and new protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this paper, the structural and functional features of these proteins in sugarcane (Saccharum officinarum L. are summarized. Since this is the first description of GRPs in sugarcane, special emphasis has been given to the expression pattern of these GRP genes by studying their abundance and prevalence in the different cDNA-libraries of the Sugarcane Expressed Sequence Tag (SUCEST project . The comparison of sugarcane GRPs with GRPs from other species is also discussed.

  12. Transcriptome analysis of the oil-rich seed of the bioenergy crop Jatropha curcas L

    Directory of Open Access Journals (Sweden)

    Moreira Raquel C

    2010-08-01

    Full Text Available Abstract Background To date, oil-rich plants are the main source of biodiesel products. Because concerns have been voiced about the impact of oil-crop cultivation on the price of food commodities, the interest in oil plants not used for food production and amenable to cultivation on non-agricultural land has soared. As a non-food, drought-resistant and oil-rich crop, Jatropha curcas L. fulfils many of the requirements for biofuel production. Results We have generated 13,249 expressed sequence tags (ESTs from developing and germinating Jatropha seeds. This strategy allowed us to detect most known genes related to lipid synthesis and degradation. We have also identified ESTs coding for proteins that may be involved in the toxicity of Jatropha seeds. Another unexpected finding is the high number of ESTs containing transposable element-related sequences in the developing seed library (800 when contrasted with those found in the germinating seed library (80. Conclusions The sequences generated in this work represent a considerable increase in the number of sequences deposited in public databases. These results can be used to produce genetically improved varieties of Jatropha with increased oil yields, different oil compositions and better agronomic characteristics.

  13. SMRT sequencing data for Garcinia mangostana L. variety Mesta

    Directory of Open Access Journals (Sweden)

    Mohd Razik Midin

    2017-06-01

    Full Text Available The “Queen of Fruits” mangosteen (Garcinia mangostana L. produces commercially important fruits with desirable taste of flesh and pericarp rich in xanthones with medicinal properties. To date, only limited knowledge is available on the cytogenetics and genome sequences of a common variety of mangosteen (Abu Bakar et al., 2016 [1]. Here, we report the first single-molecule real-time (SMRT sequencing data from whole genome sequencing of mangosteen of Mesta variety. Raw reads of the SMRT sequencing project can be obtained from SRA database with the accession numbers SRX2718652 until SRX2718659.

  14. Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

    Science.gov (United States)

    Zhang, Xiaorong

    2009-01-01

    This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR…

  15. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  16. Sequencing Intractable DNA to Close Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Hurt, Jr., Richard Ashley [ORNL; Brown, Steven D [ORNL; Podar, Mircea [ORNL; Palumbo, Anthony Vito [ORNL; Elias, Dwayne A [ORNL

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled intractable resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such difficult regions in the non-contiguous finished Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. These developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  17. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity

    Science.gov (United States)

    Simon, Joseph R.; Carroll, Nick J.; Rubinstein, Michael; Chilkoti, Ashutosh; López, Gabriel P.

    2017-06-01

    Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

  18. Computationally Efficient Chaotic Spreading Sequence Selection for Asynchronous DS-CDMA

    Directory of Open Access Journals (Sweden)

    Litviņenko Anna

    2017-12-01

    Full Text Available The choice of the spreading sequence for asynchronous direct-sequence code-division multiple-access (DS-CDMA systems plays a crucial role for the mitigation of multiple-access interference. Considering the rich dynamics of chaotic sequences, their use for spreading allows overcoming the limitations of the classical spreading sequences. However, to ensure low cross-correlation between the sequences, careful selection must be performed. This paper presents a novel exhaustive search algorithm, which allows finding sets of chaotic spreading sequences of required length with a particularly low mutual cross-correlation. The efficiency of the search is verified by simulations, which show a significant advantage compared to non-selected chaotic sequences. Moreover, the impact of sequence length on the efficiency of the selection is studied.

  19. Elucidation of the sequence selective binding mode of the DNA minor groove binder adozelesin, by high-field 1H NMR and restrained molecular dynamics

    International Nuclear Information System (INIS)

    Cameron, L.

    1999-01-01

    Adozelesin (formerly U73-975, The Upjohn Co.) is a covalent, minor-groove binding analogue of the antitumour antibiotic (+)CC-1065. Adozelesin consists of a cyclopropapyrroloindole alkylating sub-unit identical to (+)CC-1065, plus indole and benzofuran sub-units which replace the more complex pyrroloindole B and C sub-units, respectively, of (+)CC-1065. Adozelesin is a clinically important drug candidate, since it does not contain the ethylene bridge moieties on the B and C sub-units which are thought to be responsible for the unusual delayed hepatotoxicity exhibited by (+)CC-1065. Sequencing techniques identified two consensus sequences for adozelesin binding as p(dA) and 5'(T/A)(T/A)T-A*(C/G)G. This suggests that adozelesin spans a total of five base-pairs and shows a preference for A=T base-pair rich sequences, thus avoiding steric crowding around the exocyclic NH 2 of guanine and a wide minor groove. In this project, the covalent modification of two DNA sequences, i.e. 5'd(CGTAAGCGCTTA*CG) 2 and 5'-d(CGAAAAA*CGG)· 5'-d(CCGTTTTTCG), by adozelesin was examined by high-field NMR and restrained molecular mechanics and dynamics. Previous studies of minor groove binding drugs, using techniques as diverse as NMR, X-ray crystallography and molecular modelling, indicate that the incorporation of a guanine into the consensus sequence sterically hinders binding and, more importantly, produces a wider minor groove which is a 'slack' fit for the ligand. The aim of this investigation was to provide an insight into the sequence selective binding of adozelesin to 5'-AAAAA*CG and 5'-GCTTA*CG. The 1 H NMR data revealed that, in both cases, β-helical structure and Watson-Crick base-pairing was maintained on adduct formation. The 5'-GCTTA*CG adduct displayed significant distortion of the guanine base on the non-covalently modified strand. This distortion resulted from an amalgamation of two factors. Firstly, the presence of a strong hydrogen-bond between the amide linker of the

  20. Lymphatic transport and lymph node targeting of methotrexate-conjugated PEGylated dendrimers are enhanced by reducing the length of the drug linker or masking interactions with the injection site.

    Science.gov (United States)

    Ryan, Gemma M; McLeod, Victoria M; Mehta, Dharmini; Kelly, Brian D; Stanislawski, Pauline C; Owen, David J; Kaminskas, Lisa M; Porter, Christopher J H

    2017-11-01

    Drug conjugation to dendrimer-based delivery systems has been shown to enhance delivery to the lymphatic system after subcutaneous administration. Dendrimer interaction with components of the interstitium at the injection site, however, may prevent drainage from the injection site. The current study sought to vary the length of a linker employed to conjugate methotrexate (MTX) to a PEGylated dendrimer, in an attempt to reduce MTX interaction with interstitial binding sites and enhance lymphatic drainage. Dendrimers with shorter linkers resulted in higher lymphatic drainage, presumably via shielding of interaction sites by the PEG mantle, but were not retained in lymph nodes. Improved drainage of dendrimers with longer linkers was achieved through coadministration with dextran to mask interactions at the injection site while maintaining retention within the node. Enhanced drug exposure to the lymph node has the potential to enhance the treatment of lymph-node resident cancer metastases. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Analysis of high-throughput sequencing and annotation strategies for phage genomes.

    Directory of Open Access Journals (Sweden)

    Matthew R Henn

    Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

  2. Microencapsulation of Epoxidized Linseed Oil Liquid Cross-Linker in Poly(N-vinyl-pyrrolidone): Optimization by a Design-of-Experiments Approach

    NARCIS (Netherlands)

    Senatore, D.; Laven, J.; Benthem, van R.A.T.M.; La Camera, D.; With, de G.

    2010-01-01

    A liquid cross-linker, epoxidized linseed oil (ELO), was encapsulated in a plastic with a high glass transition temperature (poly(N-vinyl-2-pyrrolidone); PVP). The process parameters of the spray-drying employed were optimized by a Design-of-Experiments (DoE) approach. Three factors concerning both

  3. The ortho backbone amide linker (o-BAL) is an easily prepared and highly acid-labile handle for solid-phase synthesis

    DEFF Research Database (Denmark)

    Boas, Ulrik; Brask, Jesper; Christensen, J.B.

    2002-01-01

    The tris(alkoxy)benzyl backbone amide linker (BAL) has found widespread application in solid-phase synthesis. The key intermediate for preparation of para BAL (p-BAL) is 2,6-dimethoxy-4-hydroxybenzaldehyde; several reports on its synthesis have appeared. However, the ortho analogue of the handle (o...

  4. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

    Directory of Open Access Journals (Sweden)

    Charlotte Rehm

    Full Text Available In prokaryotes simple sequence repeats (SSRs with unit sizes of 1-5 nucleotides (nt are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4 structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc, Xanthomonas axonopodis pv. citri str. 306 (Xac, and Nostoc sp. strain PCC7120 (Ana. In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

  5. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

    Science.gov (United States)

    Rehm, Charlotte; Wurmthaler, Lena A; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S

    2015-01-01

    In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

  6. Controlling the prion propensity of glutamine/asparagine-rich proteins.

    Science.gov (United States)

    Paul, Kacy R; Ross, Eric D

    2015-01-01

    The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.

  7. Probing the Influence of Linker Length and Flexibility in the Design and Synthesis of New Trehalase Inhibitors

    Directory of Open Access Journals (Sweden)

    Giampiero D’Adamio

    2018-02-01

    Full Text Available This work aims to synthesize new trehalase inhibitors selective towards the insect trehalase versus the porcine trehalase, in view of their application as potentially non-toxic insecticides and fungicides. The synthesis of a new pseudodisaccharide mimetic 8, by means of a stereoselective α-glucosylation of the key pyrrolizidine intermediate 13, was accomplished. The activity of compound 8 as trehalase inhibitor towards C. riparius trehalase was evaluated and the results showed that 8 was active in the μM range and showed a good selectivity towards the insect trehalase. To reduce the overall number of synthetic steps, simpler and more flexible disaccharide mimetics 9–11 bearing a pyrrolidine nucleus instead of the pyrrolizidine core were synthesized. The biological data showed the key role of the linker chain’s length in inducing inhibitory properties, since only compounds 9 (α,β-mixture, bearing a two-carbon atom linker chain, maintained activity as trehalase inhibitors. A proper change in the glucosyl donor-protecting groups allowed the stereoselective synthesis of the β-glucoside 9β, which was active in the low micromolar range (IC50 = 0.78 μM and 12-fold more potent (and more selective than 9α towards the insect trehalase.

  8. Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin.

    Science.gov (United States)

    Zhang, Zhechun; Goldtzvik, Yonathan; Thirumalai, D

    2017-11-14

    Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the [Formula: see text] tubulin dimer.

  9. Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport

    International Nuclear Information System (INIS)

    Hempe, J.M.; Cousins, R.J.

    1991-01-01

    The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. The authors have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPCL and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient

  10. Traceless Azido Linker for the Solid-Phase Synthesis of NH-1,2,3-Triazoles via Cu-Catalyzed Azide-Alkyne Cycloaddition Reactions

    DEFF Research Database (Denmark)

    Cohrt, Anders Emil; Jensen, Jakob Feldthusen; Nielsen, Thomas Eiland

    2010-01-01

    A broadly useful acid-labile traceless azido linker for the solid-phase synthesis of NH-1,2,3-triazoles is presented. A variety of alkynes were efficiently immobilized on a range of polymeric supports by Cu(I)-mediated azide-alkyne cycloadditions. Supported triazoles showed excellent compatibility...

  11. Three-dimensional structure and cytokine distribution of platelet-rich fibrin.

    Science.gov (United States)

    Bai, Meng-Yi; Wang, Ching-Wei; Wang, Jyun-Yi; Lin, Ming-Fang; Chan, Wing P

    2017-02-01

    Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1) can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg); it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5) to low at the plasma end (p=0.19). Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

  12. Three-dimensional structure and cytokine distribution of platelet-rich fibrin

    Directory of Open Access Journals (Sweden)

    Meng-Yi Bai

    Full Text Available OBJECTIVES: Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1 can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. METHODS: Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg; it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. RESULTS: Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5 to low at the plasma end (p=0.19. Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. CONCLUSION: Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

  13. Synthesis and catalytic evaluation in the Heck reaction of deposited palladium catalysts immobilized via amide linkers and their molecular analogues

    Czech Academy of Sciences Publication Activity Database

    Semler, M.; Čejka, Jiří; Štěpnička, P.

    2014-01-01

    Roč. 227, MAY 2014 (2014), s. 207-214 ISSN 0920-5861 R&D Projects: GA ČR GA104/09/0561; GA ČR(CZ) GA13-08944S Institutional support: RVO:61388955 Keywords : deposited catalysts * palladium * amide linkers Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.893, year: 2014

  14. Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Kubas, Holger; Schaefer, Martin; Bauder-Wuest, Ulrike; Eder, Matthias; Oltmanns, Doerte [Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Haberkorn, Uwe; Mier, Walter [Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg (Germany); Eisenhut, Michael, E-mail: m.eisenhut@dkfz.d [Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

    2010-11-15

    Peptides involving the RGD motive (arginine-glycine-aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for {alpha}{sub v{beta}3} integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG{sub n}) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EG{sub n} linkers were investigated on their binding properties to cultured {alpha}{sub v{beta}3} integrin-expressing U87MG cells. Methods: We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with {sup 125}I-labeled echistatin. Results: While c(RGDyK) function is a relative weak competitor against [{sup 125}I]echistatin (K{sub i}, 329{+-}18 nM) for {alpha}{sub v{beta}3} integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K{sub i}, 64{+-}23 nM). This effect was even more pronounced with the RGD trimers (K{sub i}, 40{+-}7 nM) and tetramers (K{sub i}, 26{+-}9 nM). The introduction of EG{sub n} spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [{sup 125}I]echistatin. The EG{sub 6} group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer). Conclusion: The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [{sup 125}I]echistatin with the introduction of EG{sub n} spacers. This effect

  15. An ezrin-rich, rigid uropod-like structure directs movement of amoeboid blebbing cells.

    Science.gov (United States)

    Lorentzen, Anna; Bamber, Jeffrey; Sadok, Amine; Elson-Schwab, Ilan; Marshall, Christopher J

    2011-04-15

    Melanoma cells can switch between an elongated mesenchymal-type and a rounded amoeboid-type migration mode. The rounded 'amoeboid' form of cell movement is driven by actomyosin contractility resulting in membrane blebbing. Unlike elongated A375 melanoma cells, rounded A375 cells do not display any obvious morphological front-back polarisation, although polarisation is thought to be a prerequisite for cell movement. We show that blebbing A375 cells are polarised, with ezrin (a linker between the plasma membrane and actin cytoskeleton), F-actin, myosin light chain, plasma membrane, phosphatidylinositol (4,5)-bisphosphate and β1-integrin accumulating at the cell rear in a uropod-like structure. This structure does not have the typical protruding shape of classical leukocyte uropods, but, as for those structures, it is regulated by protein kinase C. We show that the ezrin-rich uropod-like structure (ERULS) is an inherent feature of polarised A375 cells and not a consequence of cell migration, and is necessary for cell invasion. Furthermore, we demonstrate that membrane blebbing is reduced at this site, leading to a model in which the rigid ezrin-containing structure determines the direction of a moving cell through localised inhibition of membrane blebbing.

  16. Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif.

    Science.gov (United States)

    Papanikolopoulou, Katerina; Teixeira, Susana; Belrhali, Hassan; Forsyth, V Trevor; Mitraki, Anna; van Raaij, Mark J

    2004-09-03

    Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid. In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation. We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon. Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues. X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A. The foldon residues 458-483 also adopt their natural structure. The intervening linkers are not well ordered in the crystal structures. This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif. It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins. Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure. They can also prove useful for gene therapy and fibre engineering applications.

  17. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    International Nuclear Information System (INIS)

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-01-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems

  18. The role of H1 linker histone subtypes in preserving the fidelity of elaboration of mesendodermal and neuroectodermal lineages during embryonic development.

    Directory of Open Access Journals (Sweden)

    Giang D Nguyen

    Full Text Available H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs depleted of H1c, H1d and H1e subtypes (H1-KO ESCs by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers.

  19. Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

    Directory of Open Access Journals (Sweden)

    Bibiana G Crespo

    Full Text Available High-throughput sequencing (HTS techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample. Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568-1,669 (95% Credible Interval and 5,027-5,196 for surface and deep water samples respectively, implying that 84-89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

  20. Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

    Science.gov (United States)

    Crespo, Bibiana G; Wallhead, Philip J; Logares, Ramiro; Pedrós-Alió, Carlos

    2016-01-01

    High-throughput sequencing (HTS) techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample). Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568-1,669 (95% Credible Interval) and 5,027-5,196 for surface and deep water samples respectively, implying that 84-89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

  1. Sequential assignment of proline-rich regions in proteins: Application to modular binding domain complexes

    International Nuclear Information System (INIS)

    Kanelis, Voula; Donaldson, Logan; Muhandiram, D.R.; Rotin, Daniela; Forman-Kay, Julie D.; Kay, Lewis E.

    2000-01-01

    Many protein-protein interactions involve amino acid sequences containing proline-rich motifs and even poly-proline stretches. The lack of amide protons in such regions complicates assignment, since 1 HN-based triple-resonance assignment strategies cannot be employed. Two such systems that we are currently studying include an SH2 domain from the protein Crk with a region containing 9 prolines in a 14 amino acid sequence, as well as a WW domain that interacts with a proline-rich target. A modified version of the HACAN pulse scheme, originally described by Bax and co-workers [Wang et al. (1995) J. Biomol. NMR, 5, 376-382], and an experiment which correlates the intra-residue 1 H α , 13 C α / 13 C β chemical shifts with the 15 N shift of the subsequent residue are presented and applied to the two systems listed above, allowing sequential assignment of the molecules

  2. Extrinsic Sources of Scatter in the Richness-Mass Relation of Galaxy Clusters

    Energy Technology Data Exchange (ETDEWEB)

    Rozo, Eduardo; /Chicago U. /Chicago U., KICP; Rykoff, Eli; /LBL, Berkeley; Koester, Benjamin; /Chicago U., Astron. Astrophys. Ctr.; Nord, Brian; /Michigan U.; Wu, Hao-Yi; /KIPAC, Menlo Park /Stanford U., Phys. Dept.; Evrard, August; /Michigan U.; Wechsler, Risa; /KIPAC, Menlo Park /Stanford U., Phys. Dept.

    2012-03-27

    Maximizing the utility of upcoming photometric cluster surveys requires a thorough understanding of the richness-mass relation of galaxy clusters. We use Monte Carlo simulations to study the impact of various sources of observational scatter on this relation. Cluster ellipticity, photometric errors, photometric redshift errors, and cluster-to-cluster variations in the properties of red-sequence galaxies contribute negligible noise. Miscentering, however, can be important, and likely contributes to the scatter in the richness - mass relation of galaxy maxBCG clusters at the low mass end, where centering is more difficult. We also investigate the impact of projection effects under several empirically motivated assumptions about cluster environments. Using SDSS data and the maxBCG cluster catalog, we demonstrate that variations in cluster environments can rarely ({approx} 1%-5% of the time) result in significant richness boosts. Due to the steepness of the mass/richness function, the corresponding fraction of optically selected clusters that suffer from these projection effects is {approx} 5%-15%. We expect these numbers to be generic in magnitude, but a precise determination requires detailed, survey-specific modeling.

  3. EXTRINSIC SOURCES OF SCATTER IN THE RICHNESS-MASS RELATION OF GALAXY CLUSTERS

    International Nuclear Information System (INIS)

    Rozo, Eduardo; Koester, Benjamin; Rykoff, Eli; Nord, Brian; Evrard, August; Wu Haoyi; Wechsler, Risa

    2011-01-01

    Maximizing the utility of upcoming photometric cluster surveys requires a thorough understanding of the richness-mass relation of galaxy clusters. We use Monte Carlo simulations to study the impact of various sources of observational scatter on this relation. Cluster ellipticity, photometric errors, photometric redshift errors, and cluster-to-cluster variations in the properties of red-sequence galaxies contribute negligible noise. Miscentering, however, can be important, and likely contributes to the scatter in the richness-mass relation of galaxy maxBCG clusters at the low-mass end, where centering is more difficult. We also investigate the impact of projection effects under several empirically motivated assumptions about cluster environments. Using Sloan Digital Sky Survey data and the maxBCG cluster catalog, we demonstrate that variations in cluster environments can rarely (∼1%-5% of the time) result in significant richness boosts. Due to the steepness of the mass/richness function, the corresponding fraction of optically selected clusters that suffer from these projection effects is ∼5%-15%. We expect these numbers to be generic in magnitude, but a precise determination requires detailed, survey-specific modeling.

  4. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  5. Preparation, structural analysis and bioactivity of ribonuclease A-albumin conjugate: tetra-conjugation or PEG as the linker.

    Science.gov (United States)

    Li, Chunju; Lin, Qixun; Wang, Jun; Shen, Lijuan; Ma, Guanghui; Su, Zhiguo; Hu, Tao

    2012-12-31

    Ribonuclease A (RNase A) is a therapeutic enzyme with cytotoxic action against tumor cells. Its clinical application is limited by the short half-life and insufficient stability. Conjugation of albumin can overcome the limitation, whereas dramatically decrease the enzymatic activity of RNase A. Here, three strategies were proposed to prepare the RNase A-bovine serum albumin (BSA) conjugates. R-SMCC-B (a conjugate of four RNase A attached with one BSA) and R-PEG-B (a mono-conjugate) were prepared using Sulfo-SMCC (a short bifunctional linker) and mal-PEG-NHS (a bifunctional PEG), respectively. Mal-PEG-NHS and hexadecylamine (HDA) were used to prepare the mono-conjugate, R-HDA-B, where HDA was adopted to bind BSA. The PEG linker can elongate the proximity between RNase A and BSA. In contrast, four RNase A were closely located on BSA in R-SMCC-B. R-SMCC-B showed the lowest K(m) and the highest relative enzymatic activity and k(cat)/K(m) in the three conjugates. Presumably, the tetravalent interaction of RNase A in R-SMCC-B can increase the binding affinity to its substrate. In addition, the slow release of BSA from R-HDA-B may increase the enzymatic activity of R-HDA-B. Our study is expected to provide strategies to develop protein-albumin conjugate with high therapeutic potential. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

    Science.gov (United States)

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-01-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic

  7. Optimizing the relaxivity of GdIII complexes appended to InP/ZnS quantum dots by linker tuning.

    Science.gov (United States)

    Stasiuk, Graeme J; Tamang, Sudarsan; Imbert, Daniel; Gateau, Christelle; Reiss, Peter; Fries, Pascal; Mazzanti, Marinella

    2013-06-21

    Three bimodal MRI/optical nanosized contrast agents with high per-nanoparticle relaxivity (up to 2523 mM(-1) s(-1) at 35 MHz and 932 mM(-1) s(-1) at 200 MHz) have been prepared connecting up to 115 tris-aqua Gd(III) complexes to fluorescent non-toxic InP/ZnS quantum dots. The structure of the linker has an important effect on the relaxivity of the final multimeric contrast agent.

  8. Analysis of gene expression in gecko digital adhesive pads indicates significant production of cysteine- and glycine-rich beta-keratins.

    Science.gov (United States)

    Hallahan, David L; Keiper-Hrynko, Natalie M; Shang, Tanya Q; Ganzke, Thaya S; Toni, Mattia; Dalla Valle, Luisa; Alibardi, Lorenzo

    2009-01-15

    Microscopic bristles (setae) present on digital pads permit the adhesion and climbing of geckos. Keratins of setae of the lizard Gekko gecko (Tokay gecko) were analyzed by the isolation of expressed mRNAs and by the generation of an EST library. Of the 510 sequences determined, 268 (52.9%) were unique. Of these, 14 appeared to encode alpha- and 111 beta-keratins. Within the beta-keratins, we identified five groups based on nucleotide sequence comparisons. Of these, one contained the bulk of beta-keratins, with 103 EST members. The mRNAs within this major group, together with two singlets, encoded cysteine-proline-serine-rich proteins of 10-14 kDa (Ge-cprp). One of the smaller groups of transcripts encoded slightly larger glycine-proline-serine-rich proteins, of 14-19 kDa (Ge-gprp). The remaining group consisted of smaller (9 kDa) serine-tyrosine-rich beta-keratins (Ge-strp). Thus three classes could be distinguished by amino acid sequence alignment. Exact matches for some of the peptide sequences obtained from setal proteins by ms/ms sequencing occur within several of these clones. Most of the beta-keratins were basic and contained a core-box region of two beta-strand sequences, with high homology to core-boxes present in avian scale and feather beta-keratins. Core-boxes are beta-folded regions that are likely responsible for polymerization into the beta-keratin filaments. The two deduced alpha-keratins of 52.7 kDa are both acidic, and contain the typical central rod region with some homology to mammalian and avian alpha-keratins, with variable N- and C-terminal regions. Basic beta-keratins and acidic alpha-keratins may interact electrostatically to form the resistant corneous material of setae. (c) 2008 Wiley-Liss, Inc.

  9. Structural analysis of a repetitive protein sequence motif in strepsirrhine primate amelogenin.

    Directory of Open Access Journals (Sweden)

    Rodrigo S Lacruz

    2011-03-01

    Full Text Available Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL, the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates.

  10. The First Extracellular Linker Is Important for Several Aspects of the Gating Mechanism of Human TRPA1 Channel

    Czech Academy of Sciences Publication Activity Database

    Maršáková, Lenka; Barvík, I.; Zíma, V.; Zímová, Lucie; Vlachová, Viktorie

    2017-01-01

    Roč. 10, Jan 31 (2017), č. článku 16. ISSN 1662-5099 R&D Projects: GA ČR(CZ) GA15-15839S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : TRP channel * S1-S2 linker * allyl isothiocynate * sensor module Subject RIV: FH - Neurology OBOR OECD: Neuroscience s (including psychophysiology Impact factor: 5.076, year: 2016

  11. The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9 bioavailability via inhibition and binding to cargo receptors

    DEFF Research Database (Denmark)

    Van den Steen, Philippe E; Van Aelst, Ilse; Hvidberg, Vibeke

    2006-01-01

    Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of approximately 64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with...... domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs....

  12. Expression and localization of special AT-rich sequence binding protein 2 in murine molar development and the pulp-dentin complex of human healthy teeth and teeth with pulpitis

    Science.gov (United States)

    He, Lina; Liu, Huimei; Shi, Lei; Pan, Shuang; Yang, Xu; Zhang, Lin; Niu, Yumei

    2017-01-01

    Special AT-rich sequence binding protein 2 (SATB2) is a member of the special family of AT-rich binding transcription factors and has a critical role in osteoblast differentiation and craniofacial patterning. However, the expression and distribution of SATB2 in tooth development is largely unknown. The aim of the present study was to detect the expression and distribution of SATB2 during murine molar development and, in human healthy teeth and teeth with pulpitis using immunohistochemistry. Molars were obtained from Kunming mice at embryonic day (E) 13.5, E14.5, E16.5 and E18.5, and postnatal day (P) 1, P5 and P7. In addition, 20 human teeth (10 healthy and 10 teeth with pulpitis) were obtained from young adult patients (age, 24.90±1.65 years) who were scheduled for routine extraction. Immunohistochemical analyses were performed to detect the expression and distribution of SATB2. The present results revealed that SATB2 exhibits a spatiotemporal expression pattern in murine molar development and was expressed in odontoblasts, predentin, dental pulp cells and the blood vessels in human teeth. These findings suggested that SATB2 may have an important role in odontoblast differentiation and dentin matrix mineralization during tooth development. PMID:29042940

  13. Assessing biosynthetic potential of agricultural groundwater through metagenomic sequencing: A diverse anammox community dominates nitrate-rich groundwater.

    Directory of Open Access Journals (Sweden)

    William B Ludington

    Full Text Available Climate change produces extremes in both temperature and precipitation causing increased drought severity and increased reliance on groundwater resources. Agricultural practices, which rely on groundwater, are sensitive to but also sources of contaminants, including nitrate. How agricultural contamination drives groundwater geochemistry through microbial metabolism is poorly understood.On an active cow dairy in the Central Valley of California, we sampled groundwater from three wells at depths of 4.3 m (two wells and 100 m (one well below ground surface (bgs as well as an effluent surface water lagoon that fertilizes surrounding corn fields. We analyzed the samples for concentrations of solutes, heavy metals, and USDA pathogenic bacteria of the Escherichia coli and Enterococcus groups as part of a long term groundwater monitoring study. Whole metagenome shotgun sequencing and assembly revealed taxonomic composition and metabolic potential of the community.Elevated nitrate and dissolved organic carbon occurred at 4.3m but not at 100m bgs. Metagenomics confirmed chemical observations and revealed several Planctomycete genomes, including a new Brocadiaceae lineage and a likely Planctomycetes OM190, as well novel diversity and high abundance of nano-prokaryotes from the Candidate Phyla Radiation (CPR, the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea (DPANN and the Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK superphyla. Pathway analysis suggests community interactions based on complimentary primary metabolic pathways and abundant secondary metabolite operons encoding antimicrobials and quorum sensing systems.The metagenomes show strong resemblance to activated sludge communities from a nitrogen removal reactor at a wastewater treatment plant, suggesting that natural bioremediation occurs through microbial metabolism. Elevated nitrate and rich secondary metabolite biosynthetic capacity suggest

  14. Exome Sequencing Provides Evidence of Polygenic Adaptation to a Fat-Rich Animal Diet in Indigenous Siberian Populations.

    Science.gov (United States)

    Hsieh, PingHsun; Hallmark, Brian; Watkins, Joseph; Karafet, Tatiana M; Osipova, Ludmila P; Gutenkunst, Ryan N; Hammer, Michael F

    2017-11-01

    Siberia is one of the coldest environments on Earth and has great seasonal temperature variation. Long-term settlement in northern Siberia undoubtedly required biological adaptation to severe cold stress, dramatic variation in photoperiod, and limited food resources. In addition, recent archeological studies show that humans first occupied Siberia at least 45,000 years ago; yet our understanding of the demographic history of modern indigenous Siberians remains incomplete. In this study, we use whole-exome sequencing data from the Nganasans and Yakuts to infer the evolutionary history of these two indigenous Siberian populations. Recognizing the complexity of the adaptive process, we designed a model-based test to systematically search for signatures of polygenic selection. Our approach accounts for stochasticity in the demographic process and the hitchhiking effect of classic selective sweeps, as well as potential biases resulting from recombination rate and mutation rate heterogeneity. Our demographic inference shows that the Nganasans and Yakuts diverged ∼12,000-13,000 years ago from East-Asian ancestors in a process involving continuous gene flow. Our polygenic selection scan identifies seven candidate gene sets with Siberian-specific signals. Three of these gene sets are related to diet, especially to fat metabolism, consistent with the hypothesis of adaptation to a fat-rich animal diet. Additional testing rejects the effect of hitchhiking and favors a model in which selection yields small allele frequency changes at multiple unlinked genes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. The specificity of Av3 sea anemone toxin for arthropods is determined at linker DI/SS2-S6 in the pore module of target sodium channels.

    Science.gov (United States)

    Gur Barzilai, Maya; Kahn, Roy; Regev, Noa; Gordon, Dalia; Moran, Yehu; Gurevitz, Michael

    2014-10-15

    Av3 is a peptide neurotoxin from the sea anemone Anemonia viridis that shows specificity for arthropod voltage-gated sodium channels (Navs). Interestingly, Av3 competes with a scorpion α-toxin on binding to insect Navs and similarly inhibits the inactivation process, and thus has been classified as 'receptor site-3 toxin', although the two peptides are structurally unrelated. This raises questions as to commonalities and differences in the way both toxins interact with Navs. Recently, site-3 was partly resolved for scorpion α-toxins highlighting S1-S2 and S3-S4 external linkers at the DIV voltage-sensor module and the juxtaposed external linkers at the DI pore module. To uncover channel determinants involved in Av3 specificity for arthropods, the toxin was examined on channel chimaeras constructed with the external linkers of the mammalian brain Nav1.2a, which is insensitive to Av3, in the background of the Drosophila DmNav1. This approach highlighted the role of linker DI/SS2-S6, adjacent to the channel pore, in determining Av3 specificity. Point mutagenesis at DI/SS2-S6 accompanied by functional assays highlighted Trp404 and His405 as a putative point of Av3 interaction with DmNav1. His405 conservation in arthropod Navs compared with tyrosine in vertebrate Navs may represent an ancient substitution that explains the contemporary selectivity of Av3. Trp404 and His405 localization near the membrane surface and the hydrophobic bioactive surface of Av3 suggest that the toxin possibly binds at a cleft by DI/S6. A partial overlap in receptor site-3 of both toxins nearby DI/S6 may explain their binding competition capabilities.

  16. VisRseq: R-based visual framework for analysis of sequencing data

    OpenAIRE

    Younesy, Hamid; Möller, Torsten; Lorincz, Matthew C; Karimi, Mohammad M; Jones, Steven JM

    2015-01-01

    Background Several tools have been developed to enable biologists to perform initial browsing and exploration of sequencing data. However the computational tool set for further analyses often requires significant computational expertise to use and many of the biologists with the knowledge needed to interpret these data must rely on programming experts. Results We present VisRseq, a framework for analysis of sequencing datasets that provides a computationally rich and accessible framework for ...

  17. Effect of the Linker in Terephthalate-Functionalized Conducting Redox Polymers

    International Nuclear Information System (INIS)

    Yang, Li; Huang, Xiao; Gogoll, Adolf; Strømme, Maria; Sjödin, Martin

    2016-01-01

    The combination of high capacity redox active pendent groups and conducting polymers, realized in conducting redox polymers (CRPs), provides materials with high charge storage capacity that are electronically conducting which makes CRPs attractive for electrical energy storage applications. In this report, six polythiophene and poly(3,4-ethylenedioxythiophene)(PEDOT)-based CRPs with a diethyl terephthalate unit covalently bound to the polymer chain by various linkers have been synthesized and characterized electrochemically. The effects of the choice of polymer backbone and of the nature of the link on the electrochemistry, and in particular the cycling stability of these polymers, are discussed. All CRPs show both the doping of the polymer backbone as well as the redox behavior of the pendent groups and the redox potential of the pendent groups in the CRPs is close to that of corresponding monomer, indicating insignificant interaction between the pendant and the polymer backbone. While all CRPs show various degrees of charge decay upon electrochemical redox conversion, the PEDOT-based CRPs show significantly improved stability compared to the polythiophene counterparts. Moreover, we show that by the right choice of link the cycling stability of diethyl terephthalate substituted PEDOT-based CRPs can be significantly improved.

  18. Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

    Science.gov (United States)

    Leaché, Adam D.; Chavez, Andreas S.; Jones, Leonard N.; Grummer, Jared A.; Gottscho, Andrew D.; Linkem, Charles W.

    2015-01-01

    Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

  19. Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

    Science.gov (United States)

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-04-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide

  20. Primary structure and localization of a conserved immunogenic Plasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle

    DEFF Research Database (Denmark)

    Borre, M B; Dziegiel, M; Høgh, B

    1991-01-01

    A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically...

  1. Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

    Science.gov (United States)

    Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

    2015-10-01

    Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

  2. Prediction of nucleosome positioning based on transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Xianfu Yi

    Full Text Available BACKGROUND: The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs have been suggested to play a role in nucleosome positioning in vivo. PRINCIPAL FINDINGS: Here, the minimum redundancy maximum relevance (mRMR feature selection algorithm, the nearest neighbor algorithm (NNA, and the incremental feature selection (IFS method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test. CONCLUSIONS: Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.

  3. Post-main-sequence planetary system evolution

    Science.gov (United States)

    Veras, Dimitri

    2016-01-01

    The fates of planetary systems provide unassailable insights into their formation and represent rich cross-disciplinary dynamical laboratories. Mounting observations of post-main-sequence planetary systems necessitate a complementary level of theoretical scrutiny. Here, I review the diverse dynamical processes which affect planets, asteroids, comets and pebbles as their parent stars evolve into giant branch, white dwarf and neutron stars. This reference provides a foundation for the interpretation and modelling of currently known systems and upcoming discoveries. PMID:26998326

  4. The Sequence and Analysis of Duplication Rich Human Chromosome 16

    Science.gov (United States)

    Martin, Joel; Han, Cliff; Gordon, Laurie A.; Terry, Astrid; Prabhakar, Shyam; She, Xinwei; Xie, Gary; Hellsten, Uffe; Man Chan, Yee; Altherr, Michael; Couronne, Olivier; Aerts, Andrea; Bajorek, Eva; Black, Stacey; Blumer, Heather; Branscomb, Elbert; Brown, Nancy C.; Bruno, William J.; Buckingham, Judith M.; Callen, David F.; Campbell, Connie S.; Campbell, Mary L.; Campbell, Evelyn W.; Caoile, Chenier; Challacombe, Jean F.; Chasteen, Leslie A.; Chertkov, Olga; Chi, Han C.; Christensen, Mari; Clark, Lynn M.; Cohn, Judith D.; Denys, Mirian; Detter, John C.; Dickson, Mark; Dimitrijevic-Bussod, Mira; Escobar, Julio; Fawcett, Joseph J.; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstein, David; Goodwin, Lynne A.; Grady, Deborah L.; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Hildebrand, Carl E.; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Jewett, Phillip E.; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Krawczyk, Marie-Claude; Leyba, Tina; Longmire, Jonathan L.; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Ludeman, Thom; Mark, Graham A.; Mcmurray, Kimberly L.; Meincke, Linda J.; Morgan, Jenna; Moyzis, Robert K.; Mundt, Mark O.; Munk, A. Christine; Nandkeshwar, Richard D.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Parson-Quintana, Beverly; Ramirez, Lucia; Rash, Sam; Retterer, James; Ricke, Darryl O.; Robinson, Donna L.; Rodriguez, Alex; Salamov, Asaf; Saunders, Elizabeth H.; Scott, Duncan; Shough, Timothy; Stallings, Raymond L.; Stalvey, Malinda; Sutherland, Robert D.; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Torney, David C.; Tran-Gyamfi, Mary; Tsai, Ming; Ulanovsky, Levy E.; Ustaszewska, Anna; Vo, Nu; White, P. Scott; Williams, Albert L.; Wills, Patricia L.; Wu, Jung-Rung; Wu, Kevin; Yang, Joan; DeJong, Pieter; Bruce, David; Doggett, Norman; Deaven, Larry; Schmutz, Jeremy; Grimwood, Jane; Richardson, Paul; et al.

    2004-01-01

    We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

  5. The sequence and analysis of duplication rich human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Joel; Han, Cliff; Gordon, Laurie A.; Terry, Astrid; Prabhakar, Shyam; She, Xinwei; Xie, Gary; Hellsten, Uffe; Man Chan, Yee; Altherr, Michael; Couronne, Olivier; Aerts, Andrea; Bajorek, Eva; Black, Stacey; Blumer, Heather; Branscomb, Elbert; Brown, Nancy C.; Bruno, William J.; Buckingham, Judith M.; Callen, David F.; Campbell, Connie S.; Campbell, Mary L.; Campbell, Evelyn W.; Caoile, Chenier; Challacombe, Jean F.; Chasteen, Leslie A.; Chertkov, Olga; Chi, Han C.; Christensen, Mari; Clark, Lynn M.; Cohn, Judith D.; Denys, Mirian; Detter, John C.; Dickson, Mark; Dimitrijevic-Bussod, Mira; Escobar, Julio; Fawcett, Joseph J.; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstein, David; Goodwin, Lynne A.; Grady, Deborah L.; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Hildebrand, Carl E.; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Jewett, Phillip E.; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Krawczyk, Marie-Claude; Leyba, Tina; Longmire, Jonathan L.; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Ludeman, Thom; Mark, Graham A.; Mcmurray, Kimberly L.; Meincke, Linda J.; Morgan, Jenna; Moyzis, Robert K.; Mundt, Mark O.; Munk, A. Christine; Nandkeshwar, Richard D.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Parson-Quintana, Beverly; Ramirez, Lucia; Rash, Sam; Retterer, James; Ricke, Darryl O.; Robinson, Donna L.; Rodriguez, Alex; Salamov, Asaf; Saunders, Elizabeth H.; Scott, Duncan; Shough, Timothy; Stallings, Raymond L.; Stalvey, Malinda; Sutherland, Robert D.; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Torney, David C.; Tran-Gyamfi, Mary; Tsai, Ming; Ulanovsky, Levy E.; Ustaszewska, Anna; Vo, Nu; White, P. Scott; Williams, Albert L.; Wills, Patricia L.; Wu, Jung-Rung; Wu, Kevin; Yang, Joan; DeJong, Pieter; Bruce, David; Doggett, Norman; Deaven, Larry; Schmutz, Jeremy; Grimwood, Jane; Richardson, Paul; et al.

    2004-08-01

    We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

  6. A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters

    Directory of Open Access Journals (Sweden)

    Lior Levy

    2017-10-01

    Full Text Available We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3–5 bp, positioned within 25 bp upstream of the Shine-Dalgarno (SD motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL design that mitigates sequence-related variance, as exemplified herein.

  7. Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

    Science.gov (United States)

    Zhang, Qian; Giebler, Holli A; Isaacson, Marisa K; Nyborg, Jennifer K

    2015-01-01

    In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo. These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust

  8. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination.

    Directory of Open Access Journals (Sweden)

    Samantha F Kornfeld

    Full Text Available Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as

  9. On site DNA barcoding by nanopore sequencing.

    Directory of Open Access Journals (Sweden)

    Michele Menegon

    Full Text Available Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet's biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities.

  10. Diols and anions can control the formation of an exciplex between a pyridinium boronic acid with an aryl group connected via a propylene linker.

    Science.gov (United States)

    Huang, Yan-Jun; Jiang, Yun-Bao; Bull, Steven D; Fossey, John S; James, Tony D

    2010-11-21

    The exciplex formation between a pyridinium boronic acid and phenyl group connected via a propylene linker can be monitored using fluorescence. Addition of pinacol affords a cyclic boronate ester with enhanced Lewis acidity that increases the strength of its cation-π stacking interaction causing a four-fold fluorescence enhancement.

  11. New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms.

    Science.gov (United States)

    Kodama, Roberto T; Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre K; Kitano, Eduardo S; Tashima, Alexandre K; Barna, Barbara F; Takakura, Ana Carolina; Serrano, Solange M T; Dias-Da-Silva, Wilmar; Tambourgi, Denise V; Portaro, Fernanda V

    2015-06-01

    The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Synthetic surfactant- and cross-linker-free preparation of highly stable lipid-polymer hybrid nanoparticles as potential oral delivery vehicles.

    Science.gov (United States)

    Wang, Taoran; Xue, Jingyi; Hu, Qiaobin; Zhou, Mingyong; Chang, Chao; Luo, Yangchao

    2017-06-05

    The toxicity associated with concentrated synthetic surfactants and the poor stability at gastrointestinal condition are two major constraints for practical applications of solid lipid nanoparticles (SLN) as oral delivery vehicles. In this study, a synthetic surfactant-free and cross-linker-free method was developed to fabricate effective, safe, and ultra-stable lipid-polymer hybrid nanoparticles (LPN). Bovine serum albumin (BSA) and dextran varying in molecular weights were first conjugated through Maillard reaction and the conjugates were exploited to emulsify solid lipid by a solvent diffusion and sonication method. The multilayer structure was formed by self-assembly of BSA-dextran micelles to envelope solid lipid via a pH- and heating-induced facile process with simultaneous surface deposition of pectin. The efficiency of different BSA-dextran conjugates was systematically studied to prepare LPN with the smallest size, the most homogeneous distribution and the greatest stability. The molecular interactions were characterized by Fourier transform infrared and fluorescence spectroscopies. Both nano spray drying and freeze-drying methods were tested to produce spherical and uniform pectin-coated LPN powders that were able to re-assemble nanoscale structure when redispersed in water. The results demonstrated the promise of a synthetic surfactant- and cross-linker-free technique to prepare highly stable pectin-coated LPN from all natural biomaterials as potential oral delivery vehicles.

  13. Wall to membrane linkers, stretch activated channels, and the detection of tension, voltage, temperature, auxin, and pH

    Science.gov (United States)

    Pickard, B. G.

    1992-01-01

    Introduction. The higher plant is a heterogeneous, mechanically prestressed structure continually subject to shifting forces. When a cell grows in a plant at gravitropic equilibrium, it must create localized maxima of shear in walls of neighboring cells. Such mechanical stress and strain are likely detected in a variety of ways. However, tension-sensitive ion channels are of particular interest because it appears that they are elaborately evolved for sensory function. We hypothesize that 1) the patchy patterns of high shear are focused via wall-to-membrane linkers onto the plasma membrane, where 2) they are translated by mechanosensory cation channels into corresponding patterns of high cytosolic Ca2+, which 3) initiate local enhancement of wall expansion. Further, we hypothesize that the local promotion of enhancement is achieved at least in part by local intensification of auxin transport across the plasma membrane. By implication, when an organ is asymmetrically pressed, rubbed, or bent or when it is displaced in the gravitational field, the net asymmetry of shear stress occurring across the organ would lead to asymmetric redistribution of auxin and corrective asymmetric growth. We shall describe a representative mechanosensitive Ca(2+) -selective cation channel (MCaC) with susceptibilities to xenobiotics implicating it as a force transducer in thigmo- and gravitropism. Then, we shall consider whether a putative wall-to-membrane linker (WML) could be a key feature of the molecular architecture permitting the stress distributed in the wall system to be focused on the channels.

  14. Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker

    Directory of Open Access Journals (Sweden)

    Satoshi Kawashima

    2016-01-01

    Full Text Available Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1 and acidic fish chitinase-2 (AFCase-2, in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa and SmeChiB (56 kDa, were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2 are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α8–fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

  15. Reversal in the relationship between species richness and turnover in a phytoplankton community.

    Science.gov (United States)

    Matthews, Blake; Pomati, Francesco

    2012-11-01

    Negative relationships between species richness and the rate of compositional turnover are common, suggesting that diverse communities have greater stability than depauperate ones; however, the mechanistic basis for this pattern is still widely debated. Species richness and turnover can covary either because they are mechanistically linked or because they share common environmental drivers. Few empirical studies have combined long-term changes in community composition with multiple drivers of environmental change, and so little is known about how the underlying mechanisms of species coexistence interact with changes in the mean and variability of environmental conditions. Here, we use a 33 year long time series (1976-2008) of phytoplankton community composition from Lake Zurich, to examine how environmental variation influences the relationship between richness and annual turnover. We find that the relationship between richness and annual turnover reverses midway through the time series (1992-1993), leading to a hump-shaped relationship between species richness and annual turnover. Using structural equation modeling we show that annual turnover and diversity are independently associated with different drivers of environmental change. Furthermore, we find that the observed annual sequences of community assembly give rise to rates of species accumulation that are more heterogeneous through time than expected by chance, likely owing to a high proportion of species showing significant autocorrelation and to strong positive covariation in the occurrences of species.

  16. Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6

    NARCIS (Netherlands)

    Reuß, Daniel R; Thürmer, Andrea; Daniel, Rolf; Quax, Wim J; Stülke, Jörg

    2016-01-01

    Bacillus subtilis ∆6 is a genome-reduced strain that was cured from six prophages and AT-rich islands. This strain is of great interest for biotechnological applications. Here, we announce the full-genome sequence of this strain. Interestingly, the conjugative element ICEBs1 has most likely

  17. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    Energy Technology Data Exchange (ETDEWEB)

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  18. Antibody reactivities to glutamate-rich peptides of Plasmodium falciparum parasites in humans from areas of different malaria endemicity

    DEFF Research Database (Denmark)

    Jakobsen, P.H.; Theander, T.G.; Hvid, L

    1996-01-01

    Synthetic P. falciparum peptides were evaluated as tools in epidemiological investigations of malaria. Plasma IgM and IgG antibody reactivities against synthetic peptides covering sequences of glutamate-rich protein (GLURP) and acidic-basic repeat antigen (ABRA) were measured by ELISA...

  19. A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT.

    Directory of Open Access Journals (Sweden)

    Pengfei Ding

    2015-06-01

    Full Text Available Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an "AT-pincer" motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA.

  20. Sequence and expression analysis of gaps in human chromosome 20

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan

    2012-01-01

    /or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ~99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing......The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and...... and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum...

  1. Molecular diversity of rumen bacterial communities from tannin-rich and fiber-rich forage fed domestic Sika deer (Cervus nippon) in China.

    Science.gov (United States)

    Li, Zhi Peng; Liu, Han Lu; Li, Guang Yu; Bao, Kun; Wang, Kai Ying; Xu, Chao; Yang, Yi Feng; Yang, Fu He; Wright, André-Denis G

    2013-07-08

    Sika deer (Cervus nippon) have different dietary preferences to other ruminants and are tolerant to tannin-rich plants. Because the rumen bacteria in domestic Sika deer have not been comprehensively studied, it is important to investigate its rumen bacterial population in order to understand its gut health and to improve the productivity of domestic Sika deer. The rumen bacterial diversity in domestic Sika deer (Cervus nippon) fed oak leaves- (OL group) and corn stalks-based diets (CS group) were elucidated using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE). Overall, 239 sequences were examined from the two groups, 139 clones from the OL group were assigned to 57 operational taxonomic units (OTUs) and 100 sequences from the CS group were divided into 50 OTUs. Prevotella-like sequences belonging to the phylum Bacteroidetes were the dominant bacteria in both groups (97.2% OL and 77% CS), and sequences related to Prevotella brevis were present in both groups. However, Prevotella shahii-like, Prevotella veroralis-like, Prevotella albensis-like, and Prevotella salivae-like sequences were abundant in the OL group compared to those in the CS group, while Succinivibrio dextrinosolvens-like and Prevotella ruminicola-like sequences were prevalent in the CS group. PCR-DGGE showed that bacterial communities clustered with respect to diets and the genus Prevotella was the dominant bacteria in the rumen of domestic Sika deer. However, the distribution of genus Prevotella from two groups was apparent. In addition, other fibrolytic bacteria, such as Clostridium populeti and Eubacterium cellulosolvens were found in the rumen of domestic Sika deer. The rumen of domestic Sika deer harbored unique bacteria which may represent novel species. The bacterial composition appeared to be affected by diet, and sequences related to Prevotella spp. may represent new species that may be related to the degradation of fiber biomass or tannins. Moreover, the mechanism

  2. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

    Science.gov (United States)

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-03-22

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

  3. Geoseq: a tool for dissecting deep-sequencing datasets

    Directory of Open Access Journals (Sweden)

    Homann Robert

    2010-10-01

    Full Text Available Abstract Background Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO, Sequence Read Archive (SRA hosted by the NCBI, or the DNA Data Bank of Japan (ddbj. Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Results Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Conclusions Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a identify differential isoform expression in mRNA-seq datasets, b identify miRNAs (microRNAs in libraries, and identify mature and star sequences in miRNAS and c to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

  4. Revealing the importance of linkers in K-series oxime reactivators for tabun-inhibited AChE using quantum chemical, docking and SMD studies.

    Science.gov (United States)

    Ghosh, Shibaji; Chandar, Nellore Bhanu; Jana, Kalyanashis; Ganguly, Bishwajit

    2017-08-01

    Inhibition of acetylcholinesterase (AChE) with organophosphorus compounds has a detrimental effect on human life. Oxime K203 seems to be one of the promising reactivators for tabun-inhibited AChE than (K027, K127, and K628). These reactivators differ only in the linker units between the two pyridinium rings. The conformational analyses performed with quantum chemical RHF/6-31G* level for K027, K127, K203 and K628 showed that the minimum energy conformers have different orientations of the active and peripheral pyridinium rings for these reactivator molecules. K203 with (-CH 2 -CH=CH-CH 2 -) linker unit possesses more open conformation compared to the other reactivators. Such orientation of K203 experiences favorable interaction with the surrounding residues of catalytic anionic site (CAS) and peripheral anionic site (PAS) of tabun-inhibited AChE. From the steered molecular dynamics simulations, it has been observed that the oxygen atom of the oxime group of K203 reactivator approaches nearest to the P-atom of the SUN203 (3.75 Å) at lower time scales (less than ~1000 ps) as compared to the other reactivators. K203 experiences less number of hydrophobic interaction with the PAS residues which is suggested to be an important factor for the efficient reactivation process. In addition, K203 crates large number of H-bonding with CAS residues SUN203, Phe295, Tyr337, Phe338 and His447. K203 barely changes its conformation during the SMD simulation process and hence the energy penalty to adopt any other conformation is minimal in this case as compared to the other reactivators. The molecular mechanics and Poisson-Boltzmann surface area binding energies obtained for the interaction of K203 inside the gorge of tabun inhibited AChE is substantially higher (-290.2 kcal/mol) than the corresponding K628 reactivator (-260.4 kcal/mol), which also possess unsaturated aromatic linker unit.

  5. Revealing the importance of linkers in K-series oxime reactivators for tabun-inhibited AChE using quantum chemical, docking and SMD studies

    Science.gov (United States)

    Ghosh, Shibaji; Chandar, Nellore Bhanu; Jana, Kalyanashis; Ganguly, Bishwajit

    2017-08-01

    Inhibition of acetylcholinesterase (AChE) with organophosphorus compounds has a detrimental effect on human life. Oxime K203 seems to be one of the promising reactivators for tabun-inhibited AChE than (K027, K127, and K628). These reactivators differ only in the linker units between the two pyridinium rings. The conformational analyses performed with quantum chemical RHF/6-31G* level for K027, K127, K203 and K628 showed that the minimum energy conformers have different orientations of the active and peripheral pyridinium rings for these reactivator molecules. K203 with (-CH2-CH=CH-CH2-) linker unit possesses more open conformation compared to the other reactivators. Such orientation of K203 experiences favorable interaction with the surrounding residues of catalytic anionic site (CAS) and peripheral anionic site (PAS) of tabun-inhibited AChE. From the steered molecular dynamics simulations, it has been observed that the oxygen atom of the oxime group of K203 reactivator approaches nearest to the P-atom of the SUN203 (3.75 Å) at lower time scales (less than 1000 ps) as compared to the other reactivators. K203 experiences less number of hydrophobic interaction with the PAS residues which is suggested to be an important factor for the efficient reactivation process. In addition, K203 crates large number of H-bonding with CAS residues SUN203, Phe295, Tyr337, Phe338 and His447. K203 barely changes its conformation during the SMD simulation process and hence the energy penalty to adopt any other conformation is minimal in this case as compared to the other reactivators. The molecular mechanics and Poisson-Boltzmann surface area binding energies obtained for the interaction of K203 inside the gorge of tabun inhibited AChE is substantially higher (-290.2 kcal/mol) than the corresponding K628 reactivator (-260.4 kcal/mol), which also possess unsaturated aromatic linker unit.

  6. An Inquiry-Oriented Approach to Span and Linear Independence: The Case of the Magic Carpet Ride Sequence

    Science.gov (United States)

    Wawro, Megan; Rasmussen, Chris; Zandieh, Michelle; Sweeney, George Franklin; Larson, Christine

    2012-01-01

    In this paper we present an innovative instructional sequence for an introductory linear algebra course that supports students' reinvention of the concepts of span, linear dependence, and linear independence. Referred to as the Magic Carpet Ride sequence, the problems begin with an imaginary scenario that allows students to build rich imagery and…

  7. Sequence and conformational preferences at termini of α-helices in membrane proteins: role of the helix environment.

    Science.gov (United States)

    Shelar, Ashish; Bansal, Manju

    2014-12-01

    α-Helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α-helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α-helices in a high-resolution dataset of integral α-helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. © 2014 Wiley Periodicals, Inc.

  8. Efficient loading of primary alcohols onto a solid phase using a trityl bromide linker

    DEFF Research Database (Denmark)

    Crestey, François; Ottesen, Lars Korsgaard; Jaroszewski, Jerzy Witold

    2008-01-01

    The Letter describes an improved, rapid and mild strategy for the loading of primary alcohols onto a polystyrene trityl resin via a highly reactive trityl bromide linker. This protocol facilitates an efficient resin loading even of acid-sensitive or heat-labile alcohols, which otherwise require...... expensive or non-commercial resin types. Secondary alcohols were only attached in moderate to low yields, while attempts to load a tertiary alcohol expectedly failed. Importantly, selective attachment of diols via a primary alcohol group in the presence of more hindered alcohol groups proved possible....... The effects of activation time and reagent excess as well as alcohol structure were investigated. This improved method provides a convenient access to O-linked resin-bound N-Fmoc-protected amino alcohols that may be employed in SPS of peptides with C-terminal alcohol functionalities. In the case...

  9. Association of poly-purine/poly-pyrimidine sequences with meiotic recombination hot spots

    Directory of Open Access Journals (Sweden)

    Pitt Joel PW

    2006-07-01

    Full Text Available Abstract Background Meiotic recombination events have been found to concentrate in 1–2.5 kilo base regions, but these recombination hot spots do not share a consensus sequence and why they occur at specific sites is not fully understood. Some previous evidence suggests that poly-purine/poly-pyrimidine (poly-pu/py tracts (PPTs, a class of sequence with distinctive biochemical properties, could be involved in recombination, but no general association of PPTs with meiotic recombination hot spots has previously been reported. Results We used computational methods to investigate in detail the relationship between PPTs and hot spots. We show statistical associations of PPT frequency with hot spots of meiotic recombination initiating lesions, double-strand breaks, in the genome of the yeast S. cerevisiae and with experimentally well characterized human meiotic recombination hot spots. Supporting a possible role of poly-pu/py-rich sequences in hot spot recombination, we also found that all three single nucleotide polymorphisms previously shown to be associated with human hot spot activity changes occur within sequence contexts of 14 bp or longer that are 85% or more poly-pu/py and at least 70% G/C. These polymorphisms are all close to the hot spot mid points. Comparing the sequences of experimentally characterized human hot spots with the orthologous regions of the chimpanzee genome previously shown not to contain hot spots, we found that in all five cases in which comparisons for the hot spot central regions are possible with publicly available sequence data, there are differences near the human hot spot mid points within sequences 14 bp or longer consisting of more than 80% poly-pu/py and at least 50% G/C. Conclusion Our results, along with previous evidence for the unique biochemical properties and recombination-stimulating potential of poly-pu/py-rich sequences, suggest that the possible functional involvement of this type of sequence in meiotic

  10. Sample limited characterization of a novel disulfide-rich venom peptide toxin from terebrid marine snail Terebra variegata.

    Directory of Open Access Journals (Sweden)

    Prachi Anand

    Full Text Available Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.

  11. Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm.

    Science.gov (United States)

    Kyne, Ciara; Crowley, Peter B

    2017-09-19

    Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1 H, 15 N HSQC and 19 F NMR spectroscopies were used to investigate the effects of C-terminal -GR n (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the "biologically inert" GB1 yields high-quality in-cell spectra, the -GR n fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR 5 motif did not improve detection of the "inert" domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nanoscale assemblies that rely on arginine-rich sequences.

  12. A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

    Science.gov (United States)

    Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

    2016-03-01

    Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

  13. Assessing the adsorption selectivity of linker functionalized, moisture-stable metal-organic framework thin films by means of an environment-controlled quartz crystal microbalance.

    Science.gov (United States)

    Bétard, Angélique; Wannapaiboon, Suttipong; Fischer, Roland A

    2012-11-04

    The stepwise thin film deposition of the robust, hydrophobic [Zn(4)O(dmcapz)(3)](n) (dmcapz = 3,5-dimethyl-4-carboxy-pyrazolato) is reported. The adsorption of small organic probe molecules, including alkanols, toluene, aniline and xylenes, was monitored by an environment-controlled quartz crystal microbalance setup. The adsorption selectivity was tuned by introducing alkyl side chains in the dmcapz linker.

  14. Funktionalisierte Linker für Metallorganische Gerüstverbindungen, deren postsynthetische Modifikation und polar markierte Schutzgruppen für terminale Alkine

    OpenAIRE

    Roy, Pascal

    2011-01-01

    Metal-organic frameworks (MOFs) form the class of porous materials with the highest surface areas. This characteristic property combined with the variability of both building blocks, the inorganic node and the organic linker, makes many scientists dream of materials with very special chemical, electronic, optic and/or magnetic properties. For individual applications both building blocks, but also possibly in the framework embedded guests, play a crucial role. This work describes the synthe...

  15. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences.

    Science.gov (United States)

    Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K; Maitra, S S

    2015-01-01

    Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about "methanogenic archaea composition" and "abundance" in the contrasting ecosystems like "landfill" and "marshland" may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

  16. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

    Directory of Open Access Journals (Sweden)

    Shailendra Yadav

    2015-01-01

    Full Text Available Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic and Thaumarchaeota (mesophilic, were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

  17. Photobiont diversity in lichens from metal-rich substrata based on ITS rDNA sequences.

    Science.gov (United States)

    Backor, Martin; Peksa, Ondrej; Skaloud, Pavel; Backorová, Miriam

    2010-05-01

    The photobiont is considered as the more sensitive partner of lichen symbiosis in metal pollution. For this reason the presence of a metal tolerant photobiont in lichens may be a key factor of ecological success of lichens growing on metal polluted substrata. The photobiont inventory was examined for terricolous lichen community growing in Cu mine-spoil heaps derived by historical mining. Sequences of internal transcribed spacer (ITS) were phylogenetically analyzed using maximum likelihood analyses. A total of 50 ITS algal sequences were obtained from 22 selected lichen taxa collected at three Cu mine-spoil heaps and two control localities. Algae associated with Cladonia and Stereocaulon were identified as members of several Asterochloris lineages, photobionts of cetrarioid lichens clustered with Trebouxia hypogymniae ined. We did not find close relationship between heavy metal content (in localities as well as lichen thalli) and photobiont diversity. Presence of multiple algal genotypes in single lichen thallus has been confirmed. Copyright 2009 Elsevier Inc. All rights reserved.

  18. The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

    Science.gov (United States)

    Sunita, S; Schwartz, Samantha L; Conn, Graeme L

    2015-11-20

    Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Micropatterning of biomolecules on a glass substrate in fused silica microchannels by using photolabile linker-based surface activation

    International Nuclear Information System (INIS)

    Jang, K.; Mawatari, K.; Kitamori, T.; Xu, Y.; Sato, K.; Tanaka, Y.

    2012-01-01

    We report on a straightforward method for creating micropatterns of multiple biomolecules. The anti-fouling agent 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and a photolabile linker (PL) were covalently linked to an amino-terminated silane surface. Patterns were generated by selective removal of the MPC polymer via UV irradiation. Multiple micropatterns of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) and rhodamine-labeled goat fragment antigen-binding fragments (FAB) were deposited on a same glass substrate. We also employed micropatterning of multiple biomolecules in that Texas red-labeled BSA and FITC-labeled rabbit anti-mouse IgG were placed inside a microchannel. (author)

  20. Unveiling Mycoplasma hyopneumoniae Promoters: Sequence Definition and Genomic Distribution

    Science.gov (United States)

    Weber, Shana de Souto; Sant'Anna, Fernando Hayashi; Schrank, Irene Silveira

    2012-01-01

    Several Mycoplasma species have had their genome completely sequenced, including four strains of the swine pathogen Mycoplasma hyopneumoniae. Nevertheless, little is known about the nucleotide sequences that control transcriptional initiation in these microorganisms. Therefore, with the objective of investigating the promoter sequences of M. hyopneumoniae, 23 transcriptional start sites (TSSs) of distinct genes were mapped. A pattern that resembles the σ70 promoter −10 element was found upstream of the TSSs. However, no −35 element was distinguished. Instead, an AT-rich periodic signal was identified. About half of the experimentally defined promoters contained the motif 5′-TRTGn-3′, which was identical to the −16 element usually found in Gram-positive bacteria. The defined promoters were utilized to build position-specific scoring matrices in order to scan putative promoters upstream of all coding sequences (CDSs) in the M. hyopneumoniae genome. Two hundred and one signals were found associated with 169 CDSs. Most of these sequences were located within 100 nucleotides of the start codons. This study has shown that the number of promoter-like sequences in the M. hyopneumoniae genome is more frequent than expected by chance, indicating that most of the sequences detected are probably biologically functional. PMID:22334569

  1. Sequencing Analysis of Mutant Allele $cdc$28-$srm$ of Protein Kinase CDC28 and Molecular Dynamics Study of Glycine-Rich Loop in Wild-Type and Mutant Allele G16S of CDK2 as Model

    CERN Document Server

    Koltovaya, N A; Kholmurodov, Kh T; Kretov, D A

    2005-01-01

    The central role that cyclin-dependent kinases play in the timing of cell division and the high incidence of genetic alteration of CDKs or deregulation of CDK inhibitors in a number of cancers make CDC28 of the yeast \\textit{Saccharomyces cerevisiae }very attractive model for studies of mechanisms of CDK regulation. Earlier it was found that certain gene mutations including \\textit{cdc28-srm} affect cell cycle progression, maintenance of different genetic structures and increase cell sensitivity to ionizing radiation. A~\\textit{cdc28-srm} mutation is not temperature-sensitive mutation and differs from the known \\textit{cdc28-ts }mutations because it has the evident phenotypic manifestations at 30 $^{\\circ}$C. Sequencing analysis of \\textit{cdc28-srm} revealed a single nucleotide substitution G20S. This is a third glycine in a conserved sequence GxGxxG in the G-rich loop positioned opposite the activation T-loop. Despite its demonstrated importance, the role of the G-loop has remained unclear. The crystal stru...

  2. Comparative analysis of chicken chromosome 28 provides new clues to the evolutionary fragility of gene-rich vertebrate regions

    NARCIS (Netherlands)

    Gordon, L.; Yang, S.; Tran-Gyamfi, M.; Baggott, D.; Christensen, M.; Hamilton, A.; Crooijmans, R.P.M.A.; Groenen, M.A.M.; Lucas, S.; Ovcharenko, I.; Stubbs, L.

    2007-01-01

    The chicken genome draft sequence has provided a valuable resource for studies of an important agricultural and experimental model species and an important data set for comparative analysis. However, some of the most gene-rich segments are missing from chicken genome draft assemblies, limiting the

  3. Optimization of micropillar sequences for fluid flow sculpting

    Energy Technology Data Exchange (ETDEWEB)

    Stoecklein, Daniel; Ganapathysubramanian, Baskar [Department of Mechanical Engineering, Iowa State University, Ames, Iowa 50011 (United States); Wu, Chueh-Yu; Kim, Donghyuk; Di Carlo, Dino [Department of Bioengineering, University of California at Los Angeles, Los Angeles, California 90095 (United States)

    2016-01-15

    Inertial fluid flow deformation around pillars in a microchannel is a new method for controlling fluid flow. Sequences of pillars have been shown to produce a rich phase space with a wide variety of flow transformations. Previous work has successfully demonstrated manual design of pillar sequences to achieve desired transformations of the flow cross section, with experimental validation. However, such a method is not ideal for seeking out complex sculpted shapes as the search space quickly becomes too large for efficient manual discovery. We explore fast, automated optimization methods to solve this problem. We formulate the inertial flow physics in microchannels with different micropillar configurations as a set of state transition matrix operations. These state transition matrices are constructed from experimentally validated streamtraces for a fixed channel length per pillar. This facilitates modeling the effect of a sequence of micropillars as nested matrix-matrix products, which have very efficient numerical implementations. With this new forward model, arbitrary micropillar sequences can be rapidly simulated with various inlet configurations, allowing optimization routines quick access to a large search space. We integrate this framework with the genetic algorithm and showcase its applicability by designing micropillar sequences for various useful transformations. We computationally discover micropillar sequences for complex transformations that are substantially shorter than manually designed sequences. We also determine sequences for novel transformations that were difficult to manually design. Finally, we experimentally validate these computational designs by fabricating devices and comparing predictions with the results from confocal microscopy.

  4. Theoretical isochrones for old, super-metal-rich stars

    International Nuclear Information System (INIS)

    Vandenberg, D.A.; Laskarides, P.G.; National Capodistrian Univ., Athens, Greece)

    1987-01-01

    A new and up-to-date set of theoretical evolutionary sequences and isochrones for super-metal-rich stars is presented. Models have been constructed for metallicities as high as Z = 0.10 and masses in the range of mass/solar mass = 0.6-1.5, in order to calculate isochrones for ages from 6 to 18 Gyr. The latest Los Alamos opacities are utilized, which have a pronounced effect on predicted mass-luminosity relations, and a value of 1.5 is adopted for the mixing-length parameter, which is required to reproduce the properties of the sun. The detailed numerical results are expected to be particularly useful in the construction of population synthesis models. 48 references

  5. MIDCOURSE SPACE EXPERIMENT VERSUS IRAS TWO-COLOR DIAGRAMS AND THE CIRCUMSTELLAR ENVELOPE-SEQUENCE OF OXYGEN-RICH LATE-TYPE STARS

    International Nuclear Information System (INIS)

    Sjouwerman, Lorant O.; Capen, Stephanie M.; Claussen, Mark J.

    2009-01-01

    We present Midcourse Space Experiment (MSX) two-color diagrams that can be used to characterize circumstellar environments of sources with good quality MSX colors in terms of IRAS color regions for oxygen-rich stars. With these diagrams, we aim to provide a new tool that can be used to study circumstellar environments and to improve detection rates for targeted surveys for circumstellar maser emission similar to the IRAS two-color diagram. This new tool is especially useful for regions in the sky where IRAS was confused, in particular in the Galactic plane and bulge region. Unfortunately, using MSX colors alone does not allow one to distinguish between carbon-rich and oxygen-rich objects. An application of this tool on 86 GHz SiO masers shows that for this type of masers an instantaneous detection rate of 60% to 80% can be achieved if target sources are selected according to MSX color (region). Our investigations may have revealed an error in the MSX point source catalog version 2.3. That is, the photometry of the 21.3 μm (MSX E filter) band for most weak 8.28 μm (or MSX A filter) band sources seems off by about a factor 2 (0.5-1 mag too bright).

  6. Mesoarchean Banded Iron Formation sequences in Dixon Island-Cleaverville Formation, Pilbara Australia: Oxygenic signal from DXCL project

    Science.gov (United States)

    Kiyokawa, S.; Ito, T.; Ikehara, M.; Yamaguchi, K. E.; Naraoka, H.; Onoue, T.; Horie, K.; Sakamoto, R.; Aihara, Y.; Miki, T.

    2013-12-01

    The 3.2-3.1 Ga Dixon island-Cleaverville formations are well-preserved Banded Iron Formation (BIF) within hydrothermal oceanic sequence at oceanic island arc setting (Kiyokawa et al., 2002, 2006, 2012). The stratigraphy of the Dixon Island (3195+15Ma) -Cleaverville (3108+13Ma) formations shows the well preserved environmental condition at the Mesoarchean ocean floor. The stratigraphy of these formations are formed about volcano-sedimentary sequences with hydrothermal chert, black shale and banded iron formation to the top. Based on the scientific drilling of DXCL project at 2007 and 2011, detail lithology between BIF sequence was clearly understood. Four drilling holes had been done at coastal sites; the Dixon Island Formation is DX site (100m) and the Cleaverville Formation is CL2 (40m), CL1 (60m) and CL3 (200m) sites and from stratigraphic bottom to top. Coarsening and thickening upward black shale-BIF sequences are well preserved of the stratigraphy form the core samples. The Dixon Island Formation consists komatiite-rhyolite sequences with many hydrothermal veins and very fine laminated cherty rocks above them. The Cleaverville Formation contains black shale, fragments-bearing pyroclastic beds, white chert, greenish shale and BIF. The CL3 core, which drilled through BIF, shows siderite-chert beds above black shale identified before magnetite lamination bed. U-Pb SHRIMP data of the tuff in lower Dixon Island Formation is 3195+15 Ma and the pyroclastic sequence below the Cleaverville BIF is 3108+13 Ma. Sedimentation rate of these sequence is 2-8 cm/ 1000year. The hole section of the organic carbon rich black shales below BIF are similar amount of organic content and 13C isotope (around -30per mill). There are very weak sulfur MIF signal (less 0.2%) in these black shale sequence. Our result show that thick organic rich sediments may be triggered to form iron rich siderite and magnetite iron beds. The stratigraphy in this sequence quite resemble to other Iron

  7. 16S rRNA-based bacterial diversity in the organic-rich sediments underlying oxygen-deficient waters of the eastern Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Divya, B.; Parvathi, A.; LokaBharathi, P.A.; Nair, S.

    ). Bootstrap analysis was carried out using 1000 iterations. Diversity analysis The clone library of AS-OMZ was compared with those of other anoxic sediments from Gulf of Mexico, North Sea and South China Sea (28, 60 and 87 sequences, respectively.... Figure 2d Bacterial diversity and richness To understand the bacterial diversity and richness of the AS-OMZ sediment clone library, comparisons were made with suboxic sediment clone libraries from South China Sea, North Sea and Gulf of Mexico...

  8. Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes

    Science.gov (United States)

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2012-01-01

    Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA–DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH–core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression. PMID:22790986

  9. Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

    Science.gov (United States)

    Kumari, Geeta; Serra, Aida; Shin, Joon; Nguyen, Phuong Q T; Sze, Siu Kwan; Yoon, Ho Sup; Tam, James P

    2015-11-25

    Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

  10. Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils.

    Directory of Open Access Journals (Sweden)

    Zewei Song

    Full Text Available Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS. The following treatments were considered: 1 the amount of soil used in DNA extraction, 2 the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation in the extraction procedure, 3 the amount of DNA template used in PCR, and 4 the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.

  11. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Science.gov (United States)

    Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

    2014-01-01

    Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  12. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  13. Deep sequencing methods for protein engineering and design.

    Science.gov (United States)

    Wrenbeck, Emily E; Faber, Matthew S; Whitehead, Timothy A

    2017-08-01

    The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering have followed. In general, these experiments address the functional consequences of thousands of protein variants in a massively parallel manner using genotype-phenotype linked high-throughput functional screens followed by DNA counting via deep sequencing. We highlight the use of information rich datasets to engineer protein molecular recognition. Examples include the creation of multiple dual-affinity Fabs targeting structurally dissimilar epitopes and engineering of a broad germline-targeted anti-HIV-1 immunogen. Additionally, we highlight the generation of enzyme fitness landscapes for conducting fundamental studies of protein behavior and evolution. We conclude with discussion of technological advances. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. A novel disulfide-rich protein motif from avian eggshell membranes.

    Directory of Open Access Journals (Sweden)

    Vamsi K Kodali

    2011-03-01

    Full Text Available Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4-C-X(5-C-X(8-C-X(6 pattern (where X represents intervening non-cysteine residues. These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata and in the oviparous green anole lizard (Anolis carolinensis. In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact

  15. Two-dimensional layer architecture assembled by Keggin polyoxotungstate, Cu(II)-EDTA complex and sodium linker: Synthesis, crystal structures, and magnetic properties

    International Nuclear Information System (INIS)

    Liu Hong; Xu Lin; Gao Guanggang; Li Fengyan; Yang Yanyan; Li Zhikui; Sun Yu

    2007-01-01

    Reaction of Keggin polyoxotungstate with copper(II)-EDTA (EDTA=ethylenediamine tetraacetate) complex under mild conditions led to the formation of hybrid inorganic-organic compounds Na 4 (OH)[(Cu 2 EDTA)PW 12 O 40 ].17H 2 O (1) and Na 4 [(Cu 2 EDTA)SiW 12 O 40 ].19H 2 O (2). The single-crystal X-ray diffraction analyses reveal their two structural features: (1) one-dimensional chain structure consisting of Keggin polyoxotungstate and copper(II)-EDTA complex; (2) Two-dimensional layer architecture assembled by the one-dimensional chain structure and sodium linker. The results of magnetic measurements in the temperature range 300-2 K indicated the existence of ferromagnetic exchange interactions between the Cu II ions for both compounds. In addition, TGA analysis, IR spectra, and electrochemical properties were also investigated to well characterize these two compounds. - Graphical abstract: Two new polyoxometalate-based hybrids, Na 4 (OH)[Cu 2 (EDTA)PW 12 O 40 ].17H 2 O (1) and Na 4 [Cu 2 (EDTA)SiW 12 O 40 ].19H 2 O (2), have been synthesized and structurally characterized, which consist of one-dimensional chain structure assembled by Keggin polyoxotungstate and copper(II)-EDTA complex. The chains are further connected to form two-dimensional layer architecture assembled by the one-dimensional chain structure and sodium linker

  16. Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation

    DEFF Research Database (Denmark)

    Wang, Zhenxing; D'Annessa, Ilda; Tesauro, Cinzia

    2015-01-01

    –DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity...... assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its...... interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of camptothecin toward the mutant....

  17. The CAPRICE RICH detector

    Energy Technology Data Exchange (ETDEWEB)

    Basini, G. [INFN, Laboratori Nazionali di Frascati, Rome (Italy); Codino, A.; Grimani, C. [Perugia Univ. (Italy)]|[INFN, Perugia (Italy); De Pascale, M.P. [Rome Univ. `Tor Vergata` (Italy). Dip. di Fisica]|[INFN, Sezione Univ. `Tor Vergata` Rome (Italy); Cafagna, F. [Bari Univ. (Italy)]|[INFN, Bari (Italy); Golden, R.L. [New Mexico State Univ., Las Cruces, NM (United States). Particle Astrophysics Lab.; Brancaccio, F.; Bocciolini, M. [Florence Univ. (Italy)]|[INFN, Florence (Italy); Barbiellini, G.; Boezio, M. [Trieste Univ. (Italy)]|[INFN, Trieste (Italy)

    1995-09-01

    A compact RICH detector has been developed and used for particle identification in a balloon borne spectrometer to measure the flux of antimatter in the cosmic radiation. This is the first RICH detector ever used in space experiments that is capable of detecting unit charged particles, such as antiprotons. The RICH and all other detectors performed well during the 27 hours long flight.

  18. The effect of linker of electrodes prepared from sol–gel ionic liquid precursor and carbon nanoparticles on dioxygen electroreduction bioelectrocatalysis

    International Nuclear Information System (INIS)

    Szot, Katarzyna; Lynch, Robert P.; Lesniewski, Adam; Majewska, Ewa; Sirieix-Plenet, Juliette; Gaillon, Laurent; Opallo, Marcin

    2011-01-01

    The effect of linker of three-dimensional, hydrophilic-carbon-nanoparticle film-electrodes prepared by layer-by-layer method on redox probe accumulation and bioelectrocatalytic dioxygen reduction was studied and compared for two different electrode scaffolds. The linker in both of these scaffolds was based on the same ionic liquid sol–gel precursor, 1-methyl-3-(3-trimethoxysilylpropyl) imidazolium bis(trifluoromethyl-sulfonyl)imide. The first electrode type was prepared by alternative immersion of tin doped indium oxide substrate in an aqueous suspension of carbon nanoparticles modified with phenyl sulphonic groups and a sol composed of ionic liquid sol–gel precursor and tetramethoxysilane. For the second electrode type sol was replaced by a methanolic suspension of silicate submicroparticles with appended imidazolium functional groups. In both films 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) anions accumulate irreversibly. In the case of the first electrode electrostatic attraction plays the more important role in comparison to the case of the second where stable adsorption of the redox probe takes place. After adsorption of bilirubin oxidase, electrodes obtained from sol and carbon nanoparticles exhibit modest bioelectrocatalytic activity towards dioxygen reduction at pH 4.8, however those obtained from oppositely charged particles are much more efficient. The magnitude of the associated catalytic current in both cases depends on the number of immersion and withdrawal steps. Interestingly, mediatorless catalysis at electrodes obtained from oppositely charged particles is more efficient than mediated catalysis.

  19. Effect of carbo-nitride-rich and oxide-rich inclusions on the pitting susceptibility of depleted uranium

    International Nuclear Information System (INIS)

    Pu, Zhen; Chen, Xianglin; Meng, Xiandong; Wu, Yanping; Shen, Liang; Wang, Qingfu; Liu, Tianwei; Shuai, Maobing

    2017-01-01

    Highlights: •The Volta potential differences relative to the matrix are positive for both types of inclusions. •Both types of inclusions are cathodic in the “inclusion/matrix” microgalvanic couples. •The oxide-rich inclusions show a larger Volta potential value of about 115 mV than the carbo-nitride-rich inclusions. •The oxide-rich inclusions give stronger local galvanic coupling with the matrix. •The oxide-rich inclusions are more predisposed to initiate pitting corrosion. -- Abstract: The effects of carbo-nitride-rich and oxide-rich inclusions on the pitting susceptibility of depleted uranium were investigated by electrochemical corrosion measurements, optical microscopy, scanning Kelvin probe force microscopy (SKPFM), and SEM. The results of the potentiodynamic polarization tests suggest that oxide-rich inclusions are more likely to induce pitting corrosion than carbo-nitride-rich inclusions. This enhanced corrosion may be explained by the strong local galvanic coupling between the oxide-rich inclusion and the surrounding matrix, which, from the sight of SKPFM analysis, exhibits a 115 V higher Volta potential than the coupling between the carbo-nitride-rich inclusions and the matrix, respectively.

  20. Proton-rich nuclear statistical equilibrium

    International Nuclear Information System (INIS)

    Seitenzahl, I.R.; Timmes, F.X.; Marin-Lafleche, A.; Brown, E.; Magkotsios, G.; Truran, J.

    2008-01-01

    Proton-rich material in a state of nuclear statistical equilibrium (NSE) is one of the least studied regimes of nucleosynthesis. One reason for this is that after hydrogen burning, stellar evolution proceeds at conditions of an equal number of neutrons and protons or at a slight degree of neutron-richness. Proton-rich nucleosynthesis in stars tends to occur only when hydrogen-rich material that accretes onto a white dwarf or a neutron star explodes, or when neutrino interactions in the winds from a nascent proto-neutron star or collapsar disk drive the matter proton-rich prior to or during the nucleosynthesis. In this Letter we solve the NSE equations for a range of proton-rich thermodynamic conditions. We show that cold proton-rich NSE is qualitatively different from neutron-rich NSE. Instead of being dominated by the Fe-peak nuclei with the largest binding energy per nucleon that have a proton-to-nucleon ratio close to the prescribed electron fraction, NSE for proton-rich material near freezeout temperature is mainly composed of 56Ni and free protons. Previous results of nuclear reaction network calculations rely on this nonintuitive high-proton abundance, which this Letter explains. We show how the differences and especially the large fraction of free protons arises from the minimization of the free energy as a result of a delicate competition between the entropy and nuclear binding energy.

  1. Digital chaotic sequence generator based on coupled chaotic systems

    International Nuclear Information System (INIS)

    Shu-Bo, Liu; Jing, Sun; Jin-Shuo, Liu; Zheng-Quan, Xu

    2009-01-01

    Chaotic systems perform well as a new rich source of cryptography and pseudo-random coding. Unfortunately their digital dynamical properties would degrade due to the finite computing precision. Proposed in this paper is a modified digital chaotic sequence generator based on chaotic logistic systems with a coupling structure where one chaotic subsystem generates perturbation signals to disturb the control parameter of the other one. The numerical simulations show that the length of chaotic orbits, the output distribution of chaotic system, and the security of chaotic sequences have been greatly improved. Moreover the chaotic sequence period can be extended at least by one order of magnitude longer than that of the uncoupled logistic system and the difficulty in decrypting increases 2 128 *2 128 times indicating that the dynamical degradation of digital chaos is effectively improved. A field programmable gate array (FPGA) implementation of an algorithm is given and the corresponding experiment shows that the output speed of the generated chaotic sequences can reach 571.4 Mbps indicating that the designed generator can be applied to the real-time video image encryption. (general)

  2. Peptidomic Identification of Cysteine-Rich Peptides from Plants.

    Science.gov (United States)

    Hemu, Xinya; Serra, Aida; Darwis, Dina A; Cornvik, Tobias; Sze, Siu Kwan; Tam, James P

    2018-01-01

    Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.

  3. Nucleosome–nucleosome interactions via histone tails and linker DNA regulate nuclear rigidity

    Science.gov (United States)

    Shimamoto, Yuta; Tamura, Sachiko; Masumoto, Hiroshi; Maeshima, Kazuhiro

    2017-01-01

    Cells, as well as the nuclei inside them, experience significant mechanical stress in diverse biological processes, including contraction, migration, and adhesion. The structural stability of nuclei must therefore be maintained in order to protect genome integrity. Despite extensive knowledge on nuclear architecture and components, however, the underlying physical and molecular mechanisms remain largely unknown. We address this by subjecting isolated human cell nuclei to microneedle-based quantitative micromanipulation with a series of biochemical perturbations of the chromatin. We find that the mechanical rigidity of nuclei depends on the continuity of the nucleosomal fiber and interactions between nucleosomes. Disrupting these chromatin features by varying cation concentration, acetylating histone tails, or digesting linker DNA results in loss of nuclear rigidity. In contrast, the levels of key chromatin assembly factors, including cohesin, condensin II, and CTCF, and a major nuclear envelope protein, lamin, are unaffected. Together with in situ evidence using living cells and a simple mechanical model, our findings reveal a chromatin-based regulation of the nuclear mechanical response and provide insight into the significance of local and global chromatin structures, such as those associated with interdigitated or melted nucleosomal fibers. PMID:28428255

  4. An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

    Science.gov (United States)

    Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

    2004-01-01

    Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

  5. Compaction of TOC-rich shales due to kerogen conversion. Implications for fluid flow and overpressure

    International Nuclear Information System (INIS)

    Hanebeck, D.; Krooss, B.M.; Leythaeuser, D.

    1998-01-01

    TOC-rich shales (10% TOC) have been artificially matured at temperatures between 200 and 350 deg C under controlled axial stress (20 - 40 MPa) for up to 350 hours. The volume change of the cylindrical samples was monitored continuously throughout the experiment. The performed experiments showed that the compaction associated with the thermal decomposition of the kerogen is significantly larger under hydrous than under dry pyrolysis conditions. This observation points at an important role of water in the conversion of kerogen. Semi quantitative permeability tests indicated that sample permeability had decreased at least one order of magnitude after the compaction pyrolysis experiments. This permeability reduction in combination with the observed compaction is the most probable mechanism for overpressure formation in TOC-rich source rock sequences. (author)

  6. DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

    2010-06-01

    A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

  7. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

    Science.gov (United States)

    Emmanuelle Morin; Annegret Kohler; Adam R. Baker; Marie Foulongne-Oriol; Vincent Lombard; Laszlo G. Nagy; Robin A. Ohm; Aleksandrina Patyshakuliyeva; Annick Brun; Andrea L. Aerts; Andrew M. Bailey; Christophe Billette; Pedro M. Coutinho; Greg Deakin; Harshavardhan Doddapaneni; Dimitrios Floudas; Jane Grimwood; Kristiina Hild& #233; n; Ursula K& #252; es; Kurt M. LaButti; Alla Lapidus; Erika A. Lindquist; Susan M. Lucas; Claude Murat; Robert W. Riley; Asaf A. Salamov; Jeremy Schmutz; Venkataramanan Subrananian; Han A.B. W& #246; sten; Jianping Xu; Daniel C. Eastwood; Gary D. Foster; Anton S.M. Sonnenberg; Daniel Cullen; Ronald P. de Vries; Taina Lundell; David S. Hibbett; Bernard Henrissat; Kerry S. Burton; Richard W. Kerrigan; Michael P. Challen; Igor V. Grigoriev; Francis Martin

    2012-01-01

    Agaricus bisporus is the model fungus for the adaptation, persistence,and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar...

  8. Platelet-rich plasma in otolaryngology.

    Science.gov (United States)

    Stavrakas, M; Karkos, P D; Markou, K; Grigoriadis, N

    2016-12-01

    Platelet-rich plasma is a novel material that is being used more frequently in many surgical specialties. A literature review on the current and potential uses of platelet-rich plasma in otolaryngology was performed. There is limited evidence on the use of platelet-rich plasma in otolaryngology compared with other specialties: only 11 studies on various subspecialties (otology, rhinology and laryngology) were included in the final review. Based on the limited number of studies, we cannot draw safe conclusions about the value of platelet-rich plasma in otolaryngology. Nevertheless, the available literature suggests that platelet-rich plasma holds promise for future research and may have a number of clinical applications.

  9. Hierarchical Self-Organization of AB n Dendron-like Molecules into a Supramolecular Lattice Sequence

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Xueyan [Department; Zhang, Ruimeng [Department; Li, Yiwen [College; Hong, You-lee [RIKEN CLST-JEOL; JEOL RESONANCE; Guo, Dong [Department; Lang, Kening [Department; Wu, Kuan-Yi [Department; Huang, Mingjun [Department; Mao, Jialin [Department; Wesdemiotis, Chrys [Department; Nishiyama, Yusuke [RIKEN CLST-JEOL; JEOL RESONANCE; Zhang, Wei [Department; Zhang, Wei [South; Miyoshi, Toshikazu [Department; Li, Tao [X-ray; Cheng, Stephen Z. D. [Department

    2017-08-07

    To understand the hierarchical self-organization behaviors of soft materials as well as their dependence on molecular geometry, a series of ABn dendron-type giant molecules based on polyhedral oligomeric silsesquioxane (POSS) nanoparticles were designed and synthesized. The apex of these molecules is a hydrophilic POSS cage with fourteen hydroxyl groups (denoted DPOSS). At its periphery, there are different numbers (n = 1–8) of hydrophobic POSS cages with seven isobutyl groups (denoted BPOSS), connected to the apical DPOSS via flexible dendron type linker(s). With varying the BPOSS number from one to seven, a supramolecular lattice formation sequence ranging from lamella (DPOSS-BPOSS), double gyroid (space group of Ia3d, DPOSS-BPOSS2), hexagonal cylinder (space group of P6mm, DPOSS-BPOSS3), Frank-Kasper A15 (space group of Pm3n, DPOSS-BPOSS4, DPOSS-BPOSS5, and DPOSS-BPOSS6), to Frank-Kasper sigma (space group of P42/mnm, DPOSS-BPOSS7) phases can be observed. The nanostructure formations in this series of ABn dendron-type giant molecules are mainly directed by the macromolecular geometric shapes. Furthermore, within each spherical motif, the soft spherical core is consisted of hydrophilic DPOSS cages with flexible linkages, while the hydrophobic BPOSS cages form the relative rigid shell and contact with neighbors to provide decreased interfaces among the spherical motifs for constructing final polyhedral motifs in these Frank-Kasper lattices. This study provides the design principle of macromolecules with specific geometric shapes and functional groups to achieve anticipated structures and macroscopic properties.

  10. Validation of rice genome sequence by optical mapping

    Directory of Open Access Journals (Sweden)

    Pape Louise

    2007-08-01

    Full Text Available Abstract Background Rice feeds much of the world, and possesses the simplest genome analyzed to date within the grass family, making it an economically relevant model system for other cereal crops. Although the rice genome is sequenced, validation and gap closing efforts require purely independent means for accurate finishing of sequence build data. Results To facilitate ongoing sequencing finishing and validation efforts, we have constructed a whole-genome SwaI optical restriction map of the rice genome. The physical map consists of 14 contigs, covering 12 chromosomes, with a total genome size of 382.17 Mb; this value is about 11% smaller than original estimates. 9 of the 14 optical map contigs are without gaps, covering chromosomes 1, 2, 3, 4, 5, 7, 8 10, and 12 in their entirety – including centromeres and telomeres. Alignments between optical and in silico restriction maps constructed from IRGSP (International Rice Genome Sequencing Project and TIGR (The Institute for Genomic Research genome sequence sources are comprehensive and informative, evidenced by map coverage across virtually all published gaps, discovery of new ones, and characterization of sequence misassemblies; all totalling ~14 Mb. Furthermore, since optical maps are ordered restriction maps, identified discordances are pinpointed on a reliable physical scaffold providing an independent resource for closure of gaps and rectification of misassemblies. Conclusion Analysis of sequence and optical mapping data effectively validates genome sequence assemblies constructed from large, repeat-rich genomes. Given this conclusion we envision new applications of such single molecule analysis that will merge advantages offered by high-resolution optical maps with inexpensive, but short sequence reads generated by emerging sequencing platforms. Lastly, map construction techniques presented here points the way to new types of comparative genome analysis that would focus on discernment of

  11. Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

    Science.gov (United States)

    Kim, Mi Kyoung; Kim, Yunyoung; Choo, Hyunah; Chong, Youhoon

    2017-02-01

    Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. An Improved Cluster Richness Estimator

    Energy Technology Data Exchange (ETDEWEB)

    Rozo, Eduardo; /Ohio State U.; Rykoff, Eli S.; /UC, Santa Barbara; Koester, Benjamin P.; /Chicago U. /KICP, Chicago; McKay, Timothy; /Michigan U.; Hao, Jiangang; /Michigan U.; Evrard, August; /Michigan U.; Wechsler, Risa H.; /SLAC; Hansen, Sarah; /Chicago U. /KICP, Chicago; Sheldon, Erin; /New York U.; Johnston, David; /Houston U.; Becker, Matthew R.; /Chicago U. /KICP, Chicago; Annis, James T.; /Fermilab; Bleem, Lindsey; /Chicago U.; Scranton, Ryan; /Pittsburgh U.

    2009-08-03

    Minimizing the scatter between cluster mass and accessible observables is an important goal for cluster cosmology. In this work, we introduce a new matched filter richness estimator, and test its performance using the maxBCG cluster catalog. Our new estimator significantly reduces the variance in the L{sub X}-richness relation, from {sigma}{sub lnL{sub X}}{sup 2} = (0.86 {+-} 0.02){sup 2} to {sigma}{sub lnL{sub X}}{sup 2} = (0.69 {+-} 0.02){sup 2}. Relative to the maxBCG richness estimate, it also removes the strong redshift dependence of the richness scaling relations, and is significantly more robust to photometric and redshift errors. These improvements are largely due to our more sophisticated treatment of galaxy color data. We also demonstrate the scatter in the L{sub X}-richness relation depends on the aperture used to estimate cluster richness, and introduce a novel approach for optimizing said aperture which can be easily generalized to other mass tracers.

  13. De novo generation of helper virus-satellite chimera RNAs results in disease attenuation and satellite sequence acquisition in a host-dependent manner.

    Science.gov (United States)

    Pyle, J D; Scholthof, Karen-Beth G

    2018-01-15

    Panicum mosaic virus (PMV) is a helper RNA virus for satellite RNAs (satRNAs) and a satellite virus (SPMV). Here, we describe modifications that occur at the 3'-end of a satRNA of PMV, satS. Co-infections of PMV+satS result in attenuation of the disease symptoms induced by PMV alone in Brachypodium distachyon and proso millet. The 375 nt satS acquires ~100-200 nts from the 3'-end of PMV during infection and is associated with decreased abundance of the PMV RNA and capsid protein in millet. PMV-satS chimera RNAs were isolated from native infections of St. Augustinegrass and switchgrass. Phylogenetic analyses revealed that the chimeric RNAs clustered according to the host species from which they were isolated. Additionally, the chimera satRNAs acquired non-viral "linker" sequences in a host-specific manner. These results highlight the dynamic regulation of viral pathogenicity by satellites, and the selective host-dependent, sequence-based pressures for driving satRNA generation and genome compositions. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

    Science.gov (United States)

    Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

    2001-08-15

    This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

  15. LHCB RICH gas system proposal

    CERN Document Server

    Bosteels, Michel; Haider, S

    2001-01-01

    Both LHCb RICH will be operated with fluorocarbon as gas radiator. RICH 1 will be filled with 4m^3 of C4F10 and RICH 2 with 100m^3 of CF4. The gas systems will run as a closed loop circulation and a gas recovery system within the closed loop is planned for RICH 1, where the recovery of the CF4 will only be realised during filling and emptying of the detector. Inline gas purification is foreseen for the gas systems in order to limit water and oxygen impurities.

  16. Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.

    Science.gov (United States)

    Allen, Cory; McDonald, Cari; Giannini, Caterina; Peng, Kah Whye; Rosales, Gabriela; Russell, Stephen J; Galanis, Evanthia

    2004-11-01

    Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP

  17. The Release 6 reference sequence of the Drosophila melanogaster genome.

    Science.gov (United States)

    Hoskins, Roger A; Carlson, Joseph W; Wan, Kenneth H; Park, Soo; Mendez, Ivonne; Galle, Samuel E; Booth, Benjamin W; Pfeiffer, Barret D; George, Reed A; Svirskas, Robert; Krzywinski, Martin; Schein, Jacqueline; Accardo, Maria Carmela; Damia, Elisabetta; Messina, Giovanni; Méndez-Lago, María; de Pablos, Beatriz; Demakova, Olga V; Andreyeva, Evgeniya N; Boldyreva, Lidiya V; Marra, Marco; Carvalho, A Bernardo; Dimitri, Patrizio; Villasante, Alfredo; Zhimulev, Igor F; Rubin, Gerald M; Karpen, Gary H; Celniker, Susan E

    2015-03-01

    Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads. © 2015 Hoskins et al.; Published by Cold Spring Harbor Laboratory Press.

  18. Analysis of the functional compatibility of SIV capsid sequences in the context of the FIV gag precursor.

    Directory of Open Access Journals (Sweden)

    César A Ovejero

    Full Text Available The formation of immature lentiviral particles is dependent on the multimerization of the Gag polyprotein at the plasma membrane of the infected cells. One key player in the virus assembly process is the capsid (CA domain of Gag, which establishes the protein-protein interactions that give rise to the hexagonal lattice of Gag molecules in the immature virion. To gain a better understanding of the functional equivalence between the CA proteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively, we generated a series of chimeric FIV Gag proteins in which the CA-coding region was partially or totally replaced by its SIV counterpart. All the FIV Gag chimeras were found to be assembly-defective; however, all of them are able to interact with wild-type SIV Gag and be recruited into extracellular virus-like particles, regardless of the SIV CA sequences present in the chimeric FIV Gag. The results presented here markedly contrast with our previous findings showing that chimeric SIVs carrying FIV CA-derived sequences are assembly-competent. Overall, our data support the notion that although the SIV and FIV CA proteins share 51% amino acid sequence similarity and exhibit a similar organization, i.e., an N-terminal domain joined by a flexible linker to a C-terminal domain, their functional exchange between these different lentiviruses is strictly dependent on the context of the recipient Gag precursor.

  19. The HADES-RICH upgrade using Hamamatsu H12700 MAPMTs with DiRICH FEE + Readout

    Science.gov (United States)

    Patel, V.; Traxler, M.

    2018-03-01

    The High Acceptance Di-Electron Spectrometer (HADES) is operational since the year 2000 and uses a hadron blind RICH detector for electron identification. The RICH photon detector is currently replaced by Hamamatsu H12700 MAPMTs with a readout system based on the DiRICH front-end module. The electronic readout chain is being developed as a joint effort of the HADES-, CBM- and PANDA collaborations and will also be used in the photon detectors for the upcoming Compressed Baryonic Matter (CBM) and PANDA experiments at FAIR . This article gives a brief overview on the photomultipliers and their quality assurance test measurements, as well as first measurements of the new DiRICH front-end module in final configurations.

  20. C-Terminally modified peptides via cleavage of the HMBA linker by O-, N- or S-nucleophiles

    DEFF Research Database (Denmark)

    Hansen, Jonas; Diness, Frederik; Meldal, Morten Peter

    2016-01-01

    A large variety of C-terminally modified peptides was obtained by nucleophilic cleavage of the ester bond in solid phase linked peptide esters of 4-hydroxymethyl benzamide (HMBA). The developed methods provided peptides, C-terminally functionalized as esters, amides and thioesters, with high purity...... directly from the resin in a single reaction step. A comprehensive screening of the reaction conditions and scope for nucleophilic cleavage of peptides from the HMBA linker was performed....

  1. Phosphonate self-assembled monolayers as organic linkers in solid-state quantum dot sensetized solar cells

    KAUST Repository

    Ardalan, Pendar

    2010-06-01

    We have employed X-ray photoelectron spectroscopy (XPS), ultraviolet-visible (UV-vis) spectroscopy, infrared (IR) spectroscopy, water contact angle (WCA) measurements, ellipsometry, and electrical measurements to study the effects of self-assembled monolayers (SAMs) with phosphonic acid headgroups on the bonding and performance of cadmium sulfide (CdS) solid-state quantum dot sensitized solar cells (QDSSCs). ∼2 to ∼6 nm size CdS quantum dots (QDs) were grown on the SAM-passivated TiO2 surfaces by successive ionic layer adsorption and reaction (SILAR). Our results show differences in the bonding of the CdS QDs at the TiO2 surfaces with a SAM linker. Moreover, our data indicate that presence of a SAM increases the CdS uptake on TiO2 as well as the performance of the resulting devices. Importantly, we observe ∼2 times higher power conversion efficiencies in the devices with a SAM compared to those that lack a SAM. © 2010 IEEE.

  2. Self-organization of Au–CdSe hybrid nanoflowers at different length scales via bi-functional diamine linkers

    Energy Technology Data Exchange (ETDEWEB)

    AbouZeid, Khaled Mohamed [Virginia Commonwealth University, Department of Chemistry (United States); Mohamed, Mona Bakr [Cairo University, National Institute of Laser Enhanced Science (NILES) (Egypt); El-Shall, M. Samy, E-mail: mselshal@vcu.edu [Virginia Commonwealth University, Department of Chemistry (United States)

    2016-01-15

    This work introduces a series of molecular bridging bi-functional linkers to produce laterally self-assembled nanostructures of the Au–CdSe nanoflowers on different length scales ranging from 10 nm to 100 microns. Assembly of Au nanocrystals within amorphous CdSe rods is found in the early stages of the growth of the Au–CdSe nanoflowers. The Au–CdSe nanoflowers are formed through a one-pot low temperature (150 °C) process where CdSe clusters are adsorbed on the surface of the Au cores, and they then start to form multiple arms and branches resulting in flower-shaped hybrid nanostructures. More complex assembly at a micron length scale can be achieved by means of bi-functional capping agents with appropriate alkyl chain lengths, such as 1,12-diaminododecane.

  3. Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155

    Directory of Open Access Journals (Sweden)

    J. Mala

    2017-06-01

    Full Text Available Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155 were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1 was confirmed by sequencing analysis. The complementarity determining regions (CDR I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

  4. A PUFA-rich diet improves fat oxidation following saturated fat-rich meal.

    Science.gov (United States)

    Stevenson, Jada L; Miller, Mary K; Skillman, Hannah E; Paton, Chad M; Cooper, Jamie A

    2017-08-01

    To determine substrate oxidation responses to saturated fatty acid (SFA)-rich meals before and after a 7-day polyunsaturated fatty acid (PUFA)-rich diet versus control diet. Twenty-six, normal-weight, adults were randomly assigned to either PUFA or control diet. Following a 3-day lead-in diet, participants completed the pre-diet visit where anthropometrics and resting metabolic rate (RMR) were measured, and two SFA-rich HF meals (breakfast and lunch) were consumed. Indirect calorimetry was used to determine fat oxidation (Fox) and energy expenditure (EE) for 4 h after each meal. Participants then consumed a PUFA-rich diet (50 % carbohydrate, 15 % protein, 35 % fat, of which 21 % of total energy was PUFA) or control diet (50 % carbohydrate, 15 % protein, 35 % fat, of which 7 % of total energy was PUFA) for the next 7 days. Following the 7-day diet, participants completed the post-diet visit. From pre- to post-PUFA-rich diet, there was no change in RMR (16.3 ± 0.8 vs. 16.4 ± 0.8 kcal/20 min) or in incremental area under the curve for EE (118.9 ± 20.6-126.9 ± 14.1 kcal/8h, ns). Fasting respiratory exchange ratio increased from pre- to post-PUFA-rich diet only (0.83 ± 0.1-0.86 ± 0.1, p diet (0.03 ± 0.1-0.23 ± 0.1 g/15 min for cumulative Fox; p diet initiates greater fat oxidation after eating occasional high SFA meals compared to a control diet, an effect achieved in 7 days.

  5. Introduction of an 8-aminooctanoic acid linker enhances uptake of 99mTc-labeled lactam bridge-cyclized α-MSH peptide in melanoma.

    Science.gov (United States)

    Guo, Haixun; Miao, Yubin

    2014-12-01

    The purpose of this study was to examine the effects of amino acid, hydrocarbon, and polyethylene glycol (PEG) linkers on the melanoma targeting and imaging properties of (99m)Tc-labeled lactam bridge-cyclized HYNIC-linker-Nle-CycMSHhex (hydrazinonicotinamide-linker-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2) peptides. Four novel peptides (HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex) were designed and synthesized. The melanocortin-1 receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The biodistribution of (99m)Tc(ethylenediaminediacetic acid [EDDA])-HYNIC-GGGNle-CycMSHhex, (99m)Tc(EDDA)-HYNIC-GSGNle-CycMSHhex, (99m)Tc(EDDA)-HYNIC-PEG2Nle-CycMSHhex, and (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h after injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were further examined because of its high melanoma uptake. The inhibitory concentrations of 50% (IC50) for HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex were 0.7 ± 0.1, 0.8 ± 0.09, 0.4 ± 0.08, and 0.3 ± 0.06 nM, respectively, in B16/F1 melanoma cells. Among these four (99m)Tc-labeled peptides, (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex displayed the highest melanoma uptake (22.3 ± 1.72 percentage injected dose/g) at 2 h after injection. (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited high tumor-to-normal-organ uptake ratios except for the kidneys. The tumor-to-kidney uptake ratios of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were 3.29, 3.63, and 6.78 at 2, 4, and 24 h, respectively, after injection. The melanoma lesions were clearly visualized by SPECT/CT using (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h after injection. High melanoma uptake and fast urinary clearance of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex highlighted its

  6. Introduction of an 8-Aminooctanoic Acid Linker Enhances the melanoma uptake of Tc-99m-labeled Lactam Bridge-Cyclized Alpha-MSH Peptide

    Science.gov (United States)

    Guo, Haixun; Miao, Yubin

    2015-01-01

    The purpose of this study was to examine the effects of amino acid, hydrocarbon and polyethylene glycol (PEG) linkers on melanoma targeting and imaging properties of 99mTc-labeled lactam bridge-cyclized HYNIC-linker-Nle-CycMSHhex {hydrazinonicotinamide-linker-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} peptides. Methods four novel peptides {HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex and HYNIC-AocNle-CycMSHhex} were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The biodistribution of 99mTc(EDDA)-HYNIC-GGGNle-CycMSHhex, 99mTc(EDDA)-HYNIC-GSGNle-CycMSHhex, 99mTc(EDDA)-HYNIC-PEG2Nle-CycMSHhex and 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h post-injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were further examined because of its high melanoma uptake. Results The IC50 values of HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex were 0.7 ± 0.1, 0.8 ± 0.09, 0.4 ± 0.08, and 0.3 ± 0.06 nM in B16/F1 melanoma cells, respectively. Among these four 99mTc-labeled peptides, 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex displayed the highest melanoma uptake (22.3 ± 1.72% ID/g) at 2 h post-injection. 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were 3.29, 3.63 and 6.78 at 2, 4 and 24 h post-injection. The melanoma lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h post-injection. Conclusion High melanoma uptake and fast urinary clearance of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex highlighted its potential for metastatic melanoma detection in the future

  7. Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: Residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family

    International Nuclear Information System (INIS)

    Contessa, Gian Marco; Orsale, Maria; Melino, Sonia; Torre, Vincent; Paci, Maurizio; Desideri, Alessandro; Cicero, Daniel O.

    2005-01-01

    The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs

  8. Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs.

    Directory of Open Access Journals (Sweden)

    Leonard T Nguyen

    2010-09-01

    Full Text Available Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the "antimicrobial centre" of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library.HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum.Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications.

  9. The constitution of alloys in the Al-rich corner of the Al-Si-Sm ternary system

    International Nuclear Information System (INIS)

    Markoli, B.; Spaic, S.; Zupanic, F.

    2001-01-01

    The constitution of alloys and the liquidus surface in the Al-rich corner of the Al-Si-Sm ternary system were determined by the examination of controlled heated and cooled specimens, as well as heat-treated specimens by means of optical and scanning electron microscopy, energy-dispersive X-ray spectroscopy, differential thermal analysis and X-ray diffraction. The Al-rich corner of the Al-Si-Sm ternary system comprises five regions of primary crystallisation (α Al , β Si , Al 3 Sm, Al 2 Si 2 Sm and AlSiSm) with following characteristic invariant reaction sequences: ternary eutectic reaction L → α Al + β Si + Al 2 Si 2 Sm, and two liquidus transition reactions, i. e., L + Al 3 Sm → α Al + AlSiSm, and L + AlSiSm → α Al + Al 2 Si 2 Sm. Along with the position of ternary eutectic and both interstitial points in the Al-rich corner of the Al-Si-Sm ternary system, the temperatures for each reaction were determined. (orig.)

  10. Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, A.K.; Huffman, D.L.; Finney, L.A.; Demeler, B.; O' Halloran, T.V.; Rosenzweig, A.C.

    2010-03-08

    PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured {Delta}({Delta}G{sup o}) {le} -8.0 kJ/mole, suggesting that copper might bind at the dimer interface.

  11. Kings Today, Rich Tomorrow

    DEFF Research Database (Denmark)

    Fattoum, Asma

    2013-01-01

    This study investigates the King vs. Rich dilemma that founder-CEOs face at IPO. When undertaking IPO, founders face two options. They can either get rich, but then run the risk of losing the control over their firms; or they can remain kings by introducing defensive mechanisms, but this is likel...

  12. The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

    International Nuclear Information System (INIS)

    Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

    2012-01-01

    Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

  13. Developments on RICH detectors

    International Nuclear Information System (INIS)

    Besson, P.; Bourgeois, P.

    1996-01-01

    The RICH (ring imaging Cherenkov) detector which is dedicated to Cherenkov radiation detection is described. An improvement made by replacing photo sensible vapor with solid photocathode is studied. A RICH detector prototype with a CsI photocathode has been built in Saclay and used with Saturne. The first results are presented. (A.C.)

  14. CAZymes Analysis Toolkit (CAT): web service for searching and analyzing carbohydrate-active enzymes in a newly sequenced organism using CAZy database.

    Science.gov (United States)

    Park, Byung H; Karpinets, Tatiana V; Syed, Mustafa H; Leuze, Michael R; Uberbacher, Edward C

    2010-12-01

    The Carbohydrate-Active Enzyme (CAZy) database provides a rich set of manually annotated enzymes that degrade, modify, or create glycosidic bonds. Despite rich and invaluable information stored in the database, software tools utilizing this information for annotation of newly sequenced genomes by CAZy families are limited. We have employed two annotation approaches to fill the gap between manually curated high-quality protein sequences collected in the CAZy database and the growing number of other protein sequences produced by genome or metagenome sequencing projects. The first approach is based on a similarity search against the entire nonredundant sequences of the CAZy database. The second approach performs annotation using links or correspondences between the CAZy families and protein family domains. The links were discovered using the association rule learning algorithm applied to sequences from the CAZy database. The approaches complement each other and in combination achieved high specificity and sensitivity when cross-evaluated with the manually curated genomes of Clostridium thermocellum ATCC 27405 and Saccharophagus degradans 2-40. The capability of the proposed framework to predict the function of unknown protein domains and of hypothetical proteins in the genome of Neurospora crassa is demonstrated. The framework is implemented as a Web service, the CAZymes Analysis Toolkit, and is available at http://cricket.ornl.gov/cgi-bin/cat.cgi.

  15. Saccharomyces cerevisiae Linker Histone Hho1p Functionally Interacts with Core Histone H4 and Negatively Regulates the Establishment of Transcriptionally Silent Chromatin*

    OpenAIRE

    Yu, Qun; Kuzmiak, Holly; Zou, Yanfei; Olsen, Lars; Defossez, Pierre-Antoine; Bi, Xin

    2009-01-01

    Saccharomyces cerevisiae linker histone Hho1p is not essential for cell viability, and very little is known about its function in vivo. We show that deletion of HHO1 (hho1Δ) suppresses the defect in transcriptional silencing caused by a mutation in the globular domain of histone H4. hho1Δ also suppresses the reduction in HML silencing by the deletion of SIR1 that is involved in the establishment of silent chromatin at HML. We further show that hho1Δ suppresses chan...

  16. Conservation and Biodiversity of Rich Fens

    DEFF Research Database (Denmark)

    Andersen, Dagmar Kappel

    2014-01-01

    Rich fen is a habitat type dependent on a constant supply of nutrient poor, calcium rich groundwater. A high, stable groundwater table, relatively high pH combined with nutrient poor conditions support a special and very species rich vegetation including many rare and threatened plant species. In...

  17. Regulating repression: roles for the sir4 N-terminus in linker DNA protection and stabilization of epigenetic states.

    Directory of Open Access Journals (Sweden)

    Stephanie Kueng

    Full Text Available Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358 must be complemented with an N-terminal domain (Sir4N; residues 1-270, expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84 derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A, which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.

  18. Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

    Directory of Open Access Journals (Sweden)

    Corentin Claeys Bouuaert

    2017-05-01

    Full Text Available Mlh1-Mlh3 (MutLγ is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ and, surprisingly, single-stranded DNA (ssDNA, which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced

  19. Is torrefaction of polysaccharides-rich biomass equivalent to carbonization of lignin-rich biomass?

    Science.gov (United States)

    Bilgic, E; Yaman, S; Haykiri-Acma, H; Kucukbayrak, S

    2016-01-01

    Waste biomass species such as lignin-rich hazelnut shell (HS) and polysaccharides-rich sunflower seed shell (SSS) were subjected to torrefaction at 300°C and carbonization at 600°C under nitrogen. The structural variations in torrefied and carbonized biomasses were compared. Also, the burning characteristics under dry air and pure oxygen (oxy-combustion) conditions were investigated. It was concluded that the effects of carbonization on HS are almost comparable with the effects of torrefaction on SSS in terms of devolatilization and deoxygenation potentials and the increases in carbon content and the heating value. Consequently, it can be proposed that torrefaction does not provide efficient devolatilization from the lignin-rich biomass while it is relatively more efficient for polysaccharides-rich biomass. Heat-induced variations in biomass led to significant changes in the burning characteristics under both burning conditions. That is, low temperature reactivity of biomass reduced considerably and the burning shifted to higher temperatures with very high burning rates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3.

    Science.gov (United States)

    Sánchez-Navarro, J A; Pallás, V

    1997-01-01

    The complete nucleotide sequence of an isolate of prunus necrotic ringspot virus (PNRSV) RNA 3 has been determined. Elucidation of the amino acid sequence of the proteins encoded by the two large open reading frames (ORFs) allowed us to carry out comparative and phylogenetic studies on the movement (MP) and coat (CP) proteins in the ilarvirus group. Amino acid sequence comparison of the MP revealed a highly conserved basic sequence motif with an amphipathic alpha-helical structure preceding the conserved motif of the '30K superfamily' proposed by Mushegian and Koonin [26] for MP's. Within this '30K' motif a strictly conserved transmembrane domain is present in all ilarviruses sequenced so far. At the amino-terminal end, prune dwarf virus (PDV) has an extension not present in other ilarviruses but which is observed in all bromo- and cucumoviruses, suggesting a common ancestor or a recombinational event in the Bromoviridae family. Examination of the N-terminus of the CP's of all ilarviruses revealed a highly basic region, part of which resembles the Arg-rich motif that has been characterized in the RNA-binding protein family. This motif has also been found in the other members of the Bromoviridae family, suggesting its involvement in a structural function. Furthermore this region is required for infectivity in ilarviruses. The similarities found in this Arg-rich motif are discussed in terms of this process known as genome activation. Finally, phylogenetic analysis of both the MP and CP proteins revealed a higher relationship of A1MV to PNRSV, apple mosaic virus (ApMV) and PDV than any other member of the ilarvirus group. In that sense, A1MV should be considered as a true ilarvirus instead of forming a distinct group of viruses.

  1. A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination

    Science.gov (United States)

    Warwood, Stacey; van Dongen, Bart; Kitchener, Andrew C.; Manning, Phillip L.

    2017-01-01

    A decade ago, reports that organic-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approximately 68 Myr Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from laboratory practice. In a subsequent study, collagen peptide sequences from an approximately 78 Myr Brachylophosaurus canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analysed by the same team, here we extract collagen from bone samples of three individuals of ostrich, Struthio camelus. The resulting LC–MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated. PMID:28566488

  2. Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

    Science.gov (United States)

    Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

    2013-01-01

    For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

  3. Combinatorial pooling enables selective sequencing of the barley gene space.

    Directory of Open Access Journals (Sweden)

    Stefano Lonardi

    2013-04-01

    Full Text Available For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

  4. Combinatorial pooling enables selective sequencing of the barley gene space.

    Science.gov (United States)

    Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

    2013-04-01

    For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

  5. Human platelet glycoprotein IX: An adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures

    International Nuclear Information System (INIS)

    Hickey, M.J.; Williams, S.A.; Roth, G.J.

    1989-01-01

    The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (M r , 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex. GPIb (M r , 165,000). Using antibody screening, the authors cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage λgt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A) + RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acids followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined

  6. General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

    International Nuclear Information System (INIS)

    Rene, Brigitte; Masliah, Gregoire; Zargarian, Loussine; Mauffret, Olivier; Fermandjian, Serge

    2006-01-01

    Summary 13 C, 15 N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments

  7. The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction.

    Science.gov (United States)

    Sundararajan, Kousik; Miguel, Amanda; Desmarais, Samantha M; Meier, Elizabeth L; Casey Huang, Kerwyn; Goley, Erin D

    2015-06-23

    The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with β-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL; however, cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment.

  8. Silica-bound copper(II)triazacyclononane as a phosphate esterase: effect of linker length and surface hydrophobicity.

    Science.gov (United States)

    Bodsgard, Brett R; Clark, Robert W; Ehrbar, Anthony W; Burstyn, Judith N

    2009-04-07

    A series of silica-bound Cu(ii) triazacyclononane materials was prepared to study the effect of linker length and surface hydrophobicity on the hydrolysis of phosphate esters. The general synthetic approach for these heterogeneous reagents was rhodium-catalyzed hydrosilation between an alkenyl-modified triazacyclononane and hydride-modified silica followed by metallation with a Cu(ii) salt. Elemental analysis confirmed that organic functionalization of the silica gel was successful and provided an estimate of the surface concentration of triazacyclononane. EPR spectra were consistent with square pyramidal Cu(ii), indicating that Cu(ii) ions were bound to the immobilized macrocycles. The hydrolytic efficacies of these heterogeneous reagents were tested with bis(p-nitrophenyl)phosphate (BNPP) and diethyl 4-nitrophenyl phosphate (paraoxon). The agent that performed best was an octyl-linked, propanol-blocked material. This material had the most hydrophilic surface and the most accessible active site, achieving a rate maximum on par with the other materials, but in fewer cycles and without an induction period.

  9. Reduction of starch granule size by expression of an engineered tandem starch-binding domain in potato plants

    NARCIS (Netherlands)

    Ji, Q.; Oomen, R.J.F.J.; Vincken, J.P.; Bolam, D.N.; Gilbert, H.J.; Suurs, L.C.J.M.; Visser, R.G.F.

    2004-01-01

    Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptice

  10. Bioaugmentation as a solution to increase methane production from an ammonia-rich substrate.

    Science.gov (United States)

    Fotidis, Ioannis A; Wang, Han; Fiedel, Nicolai R; Luo, Gang; Karakashev, Dimitar B; Angelidaki, Irini

    2014-07-01

    Ammonia-rich substrates inhibit the anaerobic digestion (AD) process and constitute the main reason for low energy recovery in full-scale reactors. It is estimated that many full-scale AD reactors are operating in ammonia induced "inhibited steady-state" with significant losses of the potential biogas production yield. To date there are not any reliable methods to alleviate the ammonia toxicity effect or to efficiently digest ammonia-rich waste. In the current study, bioaugmentation as a possible method to alleviate ammonia toxicity effect in a mesophilic continuously stirred-tank reactor (CSTR) operating under "inhibited steady state" was tested. A fast growing hydrogenotrophic methanogen (i.e., Methanoculleus bourgensis MS2(T)) was bioaugmented in the CSTR reactor at high ammonia levels (5 g NH4(+)-N L(-1)). A second CSTR reactor was used as control with no bioaugmentation. The results derived from this study clearly demonstrated a 31.3% increase in methane production yield in the CSTR reactor, at steady-state, after bioaugmentation. Additionally, high-throughput 16S rRNA gene sequencing analysis showed a 5-fold increase in relative abundance of Methanoculleus spp. after bioaugmentation. On the contrary to all methods used today to alleviate ammonia toxicity effect, the tested bioaugmentation process performed without interrupting the continuous operation of the reactor and without replacing the ammonia-rich feedstock.

  11. Realizing all reduced syzygy sequences in the planar three-body problem

    International Nuclear Information System (INIS)

    Moeckel, Richard; Montgomery, Richard

    2015-01-01

    The configuration space of the planar three-body problem, reduced by rotations and with collisions excluded, has a rich topology which supports a large set of free homotopy classes. These classes have a simple description in terms of syzygy (or eclipse) sequences. Each homotopy class corresponds to a unique ‘reduced’ syzygy sequence. We prove that each reduced syzygy sequence is realized by a periodic solution of the rotation-reduced Newtonian planar three-body problem. The realizing solutions have small, nonzero angular momentum, repeatedly come very close to triple collision, and have lots of ‘stutters’—repeated syzygies of the same type, which cancel out up to homotopy. The heart of the proof stems from the work by one of us on symbolic dynamics arising out of the central configurations after the triple collision is blown up using McGehee's method. We end with a list of open problems. (paper)

  12. A Polymorphism in the Processing Body Component Ge-1 Controls Resistance to a Naturally Occurring Rhabdovirus in Drosophila.

    Directory of Open Access Journals (Sweden)

    Chuan Cao

    2016-01-01

    Full Text Available Hosts encounter an ever-changing array of pathogens, so there is continual selection for novel ways to resist infection. A powerful way to understand how hosts evolve resistance is to identify the genes that cause variation in susceptibility to infection. Using high-resolution genetic mapping we have identified a naturally occurring polymorphism in a gene called Ge-1 that makes Drosophila melanogaster highly resistant to its natural pathogen Drosophila melanogaster sigma virus (DMelSV. By modifying the sequence of the gene in transgenic flies, we identified a 26 amino acid deletion in the serine-rich linker region of Ge-1 that is causing the resistance. Knocking down the expression of the susceptible allele leads to a decrease in viral titre in infected flies, indicating that Ge-1 is an existing restriction factor whose antiviral effects have been increased by the deletion. Ge-1 plays a central role in RNA degradation and the formation of processing bodies (P bodies. A key effector in antiviral immunity, the RNAi induced silencing complex (RISC, localises to P bodies, but we found that Ge-1-based resistance is not dependent on the small interfering RNA (siRNA pathway. However, we found that Decapping protein 1 (DCP1 protects flies against sigma virus. This protein interacts with Ge-1 and commits mRNA for degradation by removing the 5' cap, suggesting that resistance may rely on this RNA degradation pathway. The serine-rich linker domain of Ge-1 has experienced strong selection during the evolution of Drosophila, suggesting that this gene may be under long-term selection by viruses. These findings demonstrate that studying naturally occurring polymorphisms that increase resistance to infections enables us to identify novel forms of antiviral defence, and support a pattern of major effect polymorphisms controlling resistance to viruses in Drosophila.

  13. Adaptive Reactive Rich Internet Applications

    Science.gov (United States)

    Schmidt, Kay-Uwe; Stühmer, Roland; Dörflinger, Jörg; Rahmani, Tirdad; Thomas, Susan; Stojanovic, Ljiljana

    Rich Internet Applications significantly raise the user experience compared with legacy page-based Web applications because of their highly responsive user interfaces. Although this is a tremendous advance, it does not solve the problem of the one-size-fits-all approach1 of current Web applications. So although Rich Internet Applications put the user in a position to interact seamlessly with the Web application, they do not adapt to the context in which the user is currently working. In this paper we address the on-the-fly personalization of Rich Internet Applications. We introduce the concept of ARRIAs: Adaptive Reactive Rich Internet Applications and elaborate on how they are able to adapt to the current working context the user is engaged in. An architecture for the ad hoc adaptation of Rich Internet Applications is presented as well as a holistic framework and tools for the realization of our on-the-fly personalization approach. We divided both the architecture and the framework into two levels: offline/design-time and online/run-time. For design-time we explain how to use ontologies in order to annotate Rich Internet Applications and how to use these annotations for conceptual Web usage mining. Furthermore, we describe how to create client-side executable rules from the semantic data mining results. We present our declarative lightweight rule language tailored to the needs of being executed directly on the client. Because of the event-driven nature of the user interfaces of Rich Internet Applications, we designed a lightweight rule language based on the event-condition-action paradigm.2 At run-time the interactions of a user are tracked directly on the client and in real-time a user model is built up. The user model then acts as input to and is evaluated by our client-side complex event processing and rule engine.

  14. Palladium, platinum, rhodium, iridium and ruthenium in chromite- rich rocks from the Samail ophiolite, Oman.

    Science.gov (United States)

    Page, N.J.; Pallister, J.S.; Brown, M.A.; Smewing, J.D.; Haffty, J.

    1982-01-01

    30 samples of chromitite and chromite-rich rocks from two stratigraphic sections, 250 km apart, through the basal ultramafic member of the Samail ophiolite were spectrographically analysed for platinum-group elements (PGE) and for Co, Cu, Ni and V. These data are reported as are Cr/(Cr + Al), Mg/(Mg + Fe) and wt.% TiO2 for most samples. The chromitite occurs as pods or lenses in rocks of mantle origin or as discontinuous layers at the base of the overlying cumulus sequence. PGE abundances in both sections are similar, with average contents in chromite-rich rocks: Pd 8 ppb, Pt 14 ppb, Rh 6 ppb, Ir 48 ppb and Ru 135 ppb. The PGE data, combined with major-element and petrographic data on the chromitite, suggest: 1) relatively larger Ir and Ru contents and highest total PGE in the middle part of each section; 2) PGE concentrations and ratios do not correlate with coexisting silicate and chromite abundances or chromite compositions; 3) Pd/PGE, on average, increases upward in each section; 4) Samail PGE concentrations, particularly Rh, Pt and Pd, are lower than the average values for chromite-rich rocks in stratiform intrusions. 2) suggests that PGEs occur in discrete alloy or sulphide phases rather than in the major oxides or silicates, and 4) suggests that chromite-rich rocks from the oceanic upper mantle are depleted in PGE with respect to chondrites. L.C.C.

  15. Enhanced Charge Separation Efficiency in Pyridine-Anchored Phthalocyanine-Sensitized Solar Cells by Linker Elongation.

    Science.gov (United States)

    Ikeuchi, Takuro; Agrawal, Saurabh; Ezoe, Masayuki; Mori, Shogo; Kimura, Mutsumi

    2015-11-01

    A series of zinc phthalocyanine sensitizers (PcS22-24) having a pyridine anchoring group are designed and synthesized to investigate the structural dependence on performance in dye-sensitized solar cells. The pyridine-anchor zinc phthalocyanine sensitizer PcS23 shows 79 % incident-photon to current-conversion efficiency (IPCE) and 6.1 % energy conversion efficiency, which are comparable with similar phthalocyanine dyes having a carboxylic acid anchoring group. Based on DFT calculations, the high IPCE is attributed with the mixture of an excited-state molecular orbital of the sensitizer and the orbitals of TiO2 . Between pyridine and carboxylic acid anchor dyes, opposite trends are observed in the linker-length dependence of the IPCE. The red-absorbing PcS23 is applied for co-sensitization with a carboxyl-anchor organic dye D131 that has a complementary spectral response. The site-selective adsorption of PcS23 and D131 on the TiO2 surface results in a panchromatic photocurrent response for the whole visible-light region of sun light. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. The effects of alignment quality, distance calculation method, sequence filtering, and region on the analysis of 16S rRNA gene-based studies.

    Directory of Open Access Journals (Sweden)

    Patrick D Schloss

    Full Text Available Pyrosequencing of PCR-amplified fragments that target variable regions within the 16S rRNA gene has quickly become a powerful method for analyzing the membership and structure of microbial communities. This approach has revealed and introduced questions that were not fully appreciated by those carrying out traditional Sanger sequencing-based methods. These include the effects of alignment quality, the best method of calculating pairwise genetic distances for 16S rRNA genes, whether it is appropriate to filter variable regions, and how the choice of variable region relates to the genetic diversity observed in full-length sequences. I used a diverse collection of 13,501 high-quality full-length sequences to assess each of these questions. First, alignment quality had a significant impact on distance values and downstream analyses. Specifically, the greengenes alignment, which does a poor job of aligning variable regions, predicted higher genetic diversity, richness, and phylogenetic diversity than the SILVA and RDP-based alignments. Second, the effect of different gap treatments in determining pairwise genetic distances was strongly affected by the variation in sequence length for a region; however, the effect of different calculation methods was subtle when determining the sample's richness or phylogenetic diversity for a region. Third, applying a sequence mask to remove variable positions had a profound impact on genetic distances by muting the observed richness and phylogenetic diversity. Finally, the genetic distances calculated for each of the variable regions did a poor job of correlating with the full-length gene. Thus, while it is tempting to apply traditional cutoff levels derived for full-length sequences to these shorter sequences, it is not advisable. Analysis of beta-diversity metrics showed that each of these factors can have a significant impact on the comparison of community membership and structure. Taken together, these results

  17. Logo2PWM: a tool to convert sequence logo to position weight matrix.

    Science.gov (United States)

    Gao, Zhen; Liu, Lu; Ruan, Jianhua

    2017-10-03

    position weight matrix (PWM) and sequence logo are the most widely used representations of transcription factor binding site (TFBS) in biological sequences. Sequence logo - a graphical representation of PWM, has been widely used in scientific publications and reports, due to its easiness of human perception, rich information, and simple format. Different from sequence logo, PWM works great as a precise and compact digitalized form, which can be easily used by a variety of motif analysis software. There are a few available tools to generate sequence logos from PWM; however, no tool does the reverse. Such tool to convert sequence logo back to PWM is needed to scan a TFBS represented in logo format in a publication where the PWM is not provided or hard to be acquired. A major difficulty in developing such tool to convert sequence logo to PWM is to deal with the diversity of sequence logo images. We propose logo2PWM for reconstructing PWM from a large variety of sequence logo images. Evaluation results on over one thousand logos from three sources of different logo format show that the correlation between the reconstructed PWMs and the original PWMs are constantly high, where median correlation is greater than 0.97. Because of the high recognition accuracy, the easiness of usage, and, the availability of both web-based service and stand-alone application, we believe that logo2PWM can readily benefit the study of transcription by filling the gap between sequence logo and PWM.

  18. Research: Rags to Rags? Riches to Riches?

    Science.gov (United States)

    Bracey, Gerald W.

    2004-01-01

    Everyone has read about what might be called the "gold gap"--how the rich in this country are getting richer and controlling an ever-larger share of the nation's wealth. The Century Foundation has started publishing "Reality Check", a series of guides to campaign issues that sometimes finds gaps in these types of cherished delusions. The guides…

  19. Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

    Directory of Open Access Journals (Sweden)

    Matthew W. Blair

    2011-11-01

    Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

  20. The constitution of alloys in the Al-rich corner of the Al-Si-Sm ternary system

    Energy Technology Data Exchange (ETDEWEB)

    Markoli, B.; Spaic, S. [Ljubljana Univ. (Slovenia). Faculty of Natural Science and Engineering; Zupanic, F. [Maribor Univ. (Slovenia). Faculty of Mechanical Engineering

    2001-09-01

    The constitution of alloys and the liquidus surface in the Al-rich corner of the Al-Si-Sm ternary system were determined by the examination of controlled heated and cooled specimens, as well as heat-treated specimens by means of optical and scanning electron microscopy, energy-dispersive X-ray spectroscopy, differential thermal analysis and X-ray diffraction. The Al-rich corner of the Al-Si-Sm ternary system comprises five regions of primary crystallisation ({alpha}{sub Al}, {beta}{sub Si}, Al{sub 3}Sm, Al{sub 2}Si{sub 2}Sm and AlSiSm) with following characteristic invariant reaction sequences: ternary eutectic reaction L {yields} {alpha}{sub Al} + {beta}{sub Si} + Al{sub 2}Si{sub 2}Sm, and two liquidus transition reactions, i. e., L + Al{sub 3}Sm {yields} {alpha}{sub Al} + AlSiSm, and L + AlSiSm {yields} {alpha}{sub Al} + Al{sub 2}Si{sub 2}Sm. Along with the position of ternary eutectic and both interstitial points in the Al-rich corner of the Al-Si-Sm ternary system, the temperatures for each reaction were determined. (orig.)

  1. Chromium rich smectites from Jordan: a sink for hazardous elements of a high pH plume

    International Nuclear Information System (INIS)

    Khoury, H.N.

    2010-01-01

    Document available in extended abstract form only. A green earthy chromium rich di-octahedral smectite is widely distributed in Jordan. The green clay mineral is associated with marbles and travertines in central Jordan and in Suweileh area near Amman. A wide range of chromium substitution in the octahedral layer of smectites is indicated in samples from the different localities. The chromium rich smectites followed the precipitation of calcite and preceded the precipitation of opaline silica from highly alkaline paleo-groundwater. SEM photo indicating the sequence of precipitation is shown. These waters were evolved as a result of retrograde alteration and hydration reactions in the metamorphic zone. The secondary mineral precipitation followed the thermal metamorphic event of the bituminous marls. The travertine in central Jordan indicates a long-term analogue of carbonation and remobilization of silica in cementitious barriers for radioactive waste repositories. The presence of Cr-rich smectites and relatively high levels of U in the associated opaline silica may suggest the use of central Jordan outcrops as analogues with the repository disturbed zone. Smectites and silica phases are expected to be a sink for alteration products in the late stage evolution of a high pH plume. (authors)

  2. Magnetostratigraphic dating of the Xiashagou Fauna and implication for sequencing the mammalian faunas in the Nihewan Basin, North China

    NARCIS (Netherlands)

    Liu, Ping; Deng, Chenglong; Li, Shihu; Cai, Shuhui; Cheng, Hongjiang; Wei, Qi; Zhu, Rixiang

    2012-01-01

    The Nihewan Basin sedimentary sequences in northern China are rich in mammalian fossil and Paleolithic sites, thus providing insights into our understanding of Quaternary land mammal biochronology and early human settlements in East Asia. Here we present high-resolution magnetostratigraphic results

  3. Application of a continuously stirred tank bioreactor (CSTR) for bioremediation of hydrocarbon-rich industrial wastewater effluents

    International Nuclear Information System (INIS)

    Gargouri, Boutheina; Karray, Fatma; Mhiri, Najla; Aloui, Fathi; Sayadi, Sami

    2011-01-01

    A continuously stirred tank bioreactor (CSTR) was used to optimize feasible and reliable bioprocess system in order to treat hydrocarbon-rich industrial wastewaters. A successful bioremediation was developed by an efficient acclimatized microbial consortium. After an experimental period of 225 days, the process was shown to be highly efficient in decontaminating the wastewater. The performance of the bioaugmented reactor was demonstrated by the reduction of COD rates up to 95%. The residual total petroleum hydrocarbon (TPH) decreased from 320 mg TPH l -1 to 8 mg TPH l -1 . Analysis using gas chromatography-mass spectrometry (GC-MS) identified 26 hydrocarbons. The use of the mixed cultures demonstrated high degradation performance for hydrocarbons range n-alkanes (C10-C35). Six microbial isolates from the CSTR were characterized and species identification was confirmed by sequencing the 16S rRNA genes. The partial 16S rRNA gene sequences demonstrated that 5 strains were closely related to Aeromonas punctata (Aeromonas caviae), Bacillus cereus, Ochrobactrum intermedium, Stenotrophomonas maltophilia and Rhodococcus sp. The 6th isolate was affiliated to genera Achromobacter. Besides, the treated wastewater could be considered as non toxic according to the phytotoxicity test since the germination index of Lepidium sativum ranged between 57 and 95%. The treatment provided satisfactory results and presents a feasible technology for the treatment of hydrocarbon-rich wastewater from petrochemical industries and petroleum refineries.

  4. Application of a continuously stirred tank bioreactor (CSTR) for bioremediation of hydrocarbon-rich industrial wastewater effluents

    Energy Technology Data Exchange (ETDEWEB)

    Gargouri, Boutheina; Karray, Fatma; Mhiri, Najla; Aloui, Fathi [Laboratoire des Bioprocedes Environnementaux, Pole d' Excellence Regional AUF-LBPE, Centre de Biotechnologie de Sfax, Universite de Sfax, BP 1117, 3018 Sfax (Tunisia); Sayadi, Sami, E-mail: sami.sayadi@cbs.rnrt.tn [Laboratoire des Bioprocedes Environnementaux, Pole d' Excellence Regional AUF-LBPE, Centre de Biotechnologie de Sfax, Universite de Sfax, BP 1117, 3018 Sfax (Tunisia)

    2011-05-15

    A continuously stirred tank bioreactor (CSTR) was used to optimize feasible and reliable bioprocess system in order to treat hydrocarbon-rich industrial wastewaters. A successful bioremediation was developed by an efficient acclimatized microbial consortium. After an experimental period of 225 days, the process was shown to be highly efficient in decontaminating the wastewater. The performance of the bioaugmented reactor was demonstrated by the reduction of COD rates up to 95%. The residual total petroleum hydrocarbon (TPH) decreased from 320 mg TPH l{sup -1} to 8 mg TPH l{sup -1}. Analysis using gas chromatography-mass spectrometry (GC-MS) identified 26 hydrocarbons. The use of the mixed cultures demonstrated high degradation performance for hydrocarbons range n-alkanes (C10-C35). Six microbial isolates from the CSTR were characterized and species identification was confirmed by sequencing the 16S rRNA genes. The partial 16S rRNA gene sequences demonstrated that 5 strains were closely related to Aeromonas punctata (Aeromonas caviae), Bacillus cereus, Ochrobactrum intermedium, Stenotrophomonas maltophilia and Rhodococcus sp. The 6th isolate was affiliated to genera Achromobacter. Besides, the treated wastewater could be considered as non toxic according to the phytotoxicity test since the germination index of Lepidium sativum ranged between 57 and 95%. The treatment provided satisfactory results and presents a feasible technology for the treatment of hydrocarbon-rich wastewater from petrochemical industries and petroleum refineries.

  5. Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

    Science.gov (United States)

    Glynn, Neil C; Comstock, Jack C; Sood, Sushma G; Dang, Phat M; Chaparro, Jose X

    2008-01-01

    Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

  6. The soybean-Phytophthora resistance locus Rps1-k encompasses coiled coil-nucleotide binding-leucine rich repeat-like genes and repetitive sequences

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Madan K

    2008-03-01

    Full Text Available Abstract Background A series of Rps (resistance to Pytophthora sojae genes have been protecting soybean from the root and stem rot disease caused by the Oomycete pathogen, Phytophthora sojae. Five Rps genes were mapped to the Rps1 locus located near the 28 cM map position on molecular linkage group N of the composite genetic soybean map. Among these five genes, Rps1-k was introgressed from the cultivar, Kingwa. Rps1-k has been providing stable and broad-spectrum Phytophthora resistance in the major soybean-producing regions of the United States. Rps1-k has been mapped and isolated. More than one functional Rps1-k gene was identified from the Rps1-k locus. The clustering feature at the Rps1-k locus might have facilitated the expansion of Rps1-k gene numbers and the generation of new recognition specificities. The Rps1-k region was sequenced to understand the possible evolutionary steps that shaped the generation of Phytophthora resistance genes in soybean. Results Here the analyses of sequences of three overlapping BAC clones containing the 184,111 bp Rps1-k region are reported. A shotgun sequencing strategy was applied in sequencing the BAC contig. Sequence analysis predicted a few full-length genes including two Rps1-k genes, Rps1-k-1 and Rps1-k-2. Previously reported Rps1-k-3 from this genomic region 1 was evolved through intramolecular recombination between Rps1-k-1 and Rps1-k-2 in Escherichia coli. The majority of the predicted genes are truncated and therefore most likely they are nonfunctional. A member of a highly abundant retroelement, SIRE1, was identified from the Rps1-k region. The Rps1-k region is primarily composed of repetitive sequences. Sixteen simple repeat and 63 tandem repeat sequences were identified from the locus. Conclusion These data indicate that the Rps1 locus is located in a gene-poor region. The abundance of repetitive sequences in the Rps1-k region suggested that the location of this locus is in or near a

  7. A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

    Science.gov (United States)

    Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

    2016-06-01

    Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for

  8. Graph mining for next generation sequencing: leveraging the assembly graph for biological insights.

    Science.gov (United States)

    Warnke-Sommer, Julia; Ali, Hesham

    2016-05-06

    The assembly of Next Generation Sequencing (NGS) reads remains a challenging task. This is especially true for the assembly of metagenomics data that originate from environmental samples potentially containing hundreds to thousands of unique species. The principle objective of current assembly tools is to assemble NGS reads into contiguous stretches of sequence called contigs while maximizing for both accuracy and contig length. The end goal of this process is to produce longer contigs with the major focus being on assembly only. Sequence read assembly is an aggregative process, during which read overlap relationship information is lost as reads are merged into longer sequences or contigs. The assembly graph is information rich and capable of capturing the genomic architecture of an input read data set. We have developed a novel hybrid graph in which nodes represent sequence regions at different levels of granularity. This model, utilized in the assembly and analysis pipeline Focus, presents a concise yet feature rich view of a given input data set, allowing for the extraction of biologically relevant graph structures for graph mining purposes. Focus was used to create hybrid graphs to model metagenomics data sets obtained from the gut microbiomes of five individuals with Crohn's disease and eight healthy individuals. Repetitive and mobile genetic elements are found to be associated with hybrid graph structure. Using graph mining techniques, a comparative study of the Crohn's disease and healthy data sets was conducted with focus on antibiotics resistance genes associated with transposase genes. Results demonstrated significant differences in the phylogenetic distribution of categories of antibiotics resistance genes in the healthy and diseased patients. Focus was also evaluated as a pure assembly tool and produced excellent results when compared against the Meta-velvet, Omega, and UD-IDBA assemblers. Mining the hybrid graph can reveal biological phenomena captured

  9. Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

    Science.gov (United States)

    2011-01-01

    Background Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. Results De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. Conclusion This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the

  10. Mixed Media Richness and Computer-Mediated Communications

    OpenAIRE

    Atkins, Anthony B.

    2006-01-01

    Mixed richness communications occur when a participant in a conversation receives a different media or combination of media than they transmit. Mixed richness communications occur in the workplace when technical, physiological or practical limitations prevent the use of the same media on both ends of a conversation. Prior research in CMC has focused on same-richness communications, and the design guidelines that are available for same-richness communications may not be applicable to mixed-r...

  11. Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau

    Science.gov (United States)

    McFadden, C. S.; Brown, A. S.; Brayton, C.; Hunt, C. B.; van Ofwegen, L. P.

    2014-06-01

    The application of DNA barcoding to anthozoan cnidarians has been hindered by their slow rates of mitochondrial gene evolution and the failure to identify alternative molecular markers that distinguish species reliably. Among octocorals, however, multilocus barcodes can distinguish up to 70 % of morphospecies, thereby facilitating the identification of species that are ecologically important but still very poorly known taxonomically. We tested the ability of these imperfect DNA barcodes to estimate species richness in a biodiversity survey of the shallow-water octocoral fauna of Palau using multilocus ( COI, mtMutS, 28S rDNA) sequences obtained from 305 specimens representing 38 genera of octocorals. Numbers and identities of species were estimated independently (1) by a taxonomic expert using morphological criteria and (2) by assigning sequences to molecular operational taxonomic units (MOTUs) using predefined genetic distance thresholds. Estimated numbers of MOTUs ranged from 73 to 128 depending on the barcode and distance threshold applied, bracketing the estimated number of 118 morphospecies. Concordance between morphospecies identifications and MOTUs ranged from 71 to 75 % and differed little among barcodes. For the speciose and ecologically dominant genus Sinularia, however, we were able to identify 95 % of specimens correctly simply by comparing mtMutS sequences and in situ photographs of colonies to an existing vouchered database. Because we lack a clear understanding of species boundaries in most of these taxa, numbers of morphospecies and MOTUs are both estimates of the true species diversity, and we cannot currently determine which is more accurate. Our results suggest, however, that the two methods provide comparable estimates of species richness for shallow-water Indo-Pacific octocorals. Use of molecular barcodes in biodiversity surveys will facilitate comparisons of species richness and composition among localities and over time, data that do not

  12. Species richness, area and climate correlates

    DEFF Research Database (Denmark)

    Nogues, David Bravo; Bastos Araujo, Miguel

    2006-01-01

    affects: (1) the selection of climate variables entering a species richness model; and (2) the accuracy of models in predicting species richness in unsampled grid cells. Location Western Europe. Methods Models are developed for European plant, breeding bird, mammal and herptile species richness using...... seven climate variables. Generalized additive models are used to relate species richness, climate and area. Results We found that variation in the grid cell area was large (50 × 50 km: 8-3311 km2; 220 × 220: 193-55,100 km2), but this did not affect the selection of variables in the models. Similarly...... support the assumption that variation in near-equal area cells may be of second-order importance for models explaining or predicting species richness in relation to climate, although there is a possibility that drops in accuracy might increase with grid cell size. The results are, however, contingent...

  13. Evolutionary rates of mitochondrial genomes correspond to diversification rates and to contemporary species richness in birds and reptiles

    Science.gov (United States)

    Eo, Soo Hyung; DeWoody, J. Andrew

    2010-01-01

    Rates of biological diversification should ultimately correspond to rates of genome evolution. Recent studies have compared diversification rates with phylogenetic branch lengths, but incomplete phylogenies hamper such analyses for many taxa. Herein, we use pairwise comparisons of confamilial sauropsid (bird and reptile) mitochondrial DNA (mtDNA) genome sequences to estimate substitution rates. These molecular evolutionary rates are considered in light of the age and species richness of each taxonomic family, using a random-walk speciation–extinction process to estimate rates of diversification. We find the molecular clock ticks at disparate rates in different families and at different genes. For example, evolutionary rates are relatively fast in snakes and lizards, intermediate in crocodilians and slow in turtles and birds. There was also rate variation across genes, where non-synonymous substitution rates were fastest at ATP8 and slowest at CO3. Family-by-gene interactions were significant, indicating that local clocks vary substantially among sauropsids. Most importantly, we find evidence that mitochondrial genome evolutionary rates are positively correlated with speciation rates and with contemporary species richness. Nuclear sequences are poorly represented among reptiles, but the correlation between rates of molecular evolution and species diversification also extends to 18 avian nuclear genes we tested. Thus, the nuclear data buttress our mtDNA findings. PMID:20610427

  14. Cloning and sequence of the human adrenodoxin reductase gene

    International Nuclear Information System (INIS)

    Lin, Dong; Shi, Y.; Miller, W.L.

    1990-01-01

    Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

  15. Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

    Directory of Open Access Journals (Sweden)

    Ka H. Wong

    2017-06-01

    Full Text Available Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

  16. Crystal Structure of the FERM Domain of Focal Adhesion Kinase

    International Nuclear Information System (INIS)

    Ceccarelli, D.; Song, H.; Poy, F.; Schaller, M.; Eck, M.

    2006-01-01

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment

  17. A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination.

    Science.gov (United States)

    Buckley, Michael; Warwood, Stacey; van Dongen, Bart; Kitchener, Andrew C; Manning, Phillip L

    2017-05-31

    A decade ago, reports that organic-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approximately 68 Myr Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from laboratory practice. In a subsequent study, collagen peptide sequences from an approximately 78 Myr Brachylophosaurus canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analysed by the same team, here we extract collagen from bone samples of three individuals of ostrich, Struthio camelus The resulting LC-MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated. © 2017 The Authors.

  18. 454 sequencing of pooled BAC clones on chromosome 3H of barley

    Directory of Open Access Journals (Sweden)

    Yamaji Nami

    2011-05-01

    Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

  19. Reagents for Astatination of Biomolecules. 5. Evaluation of hydrazone linkers in 211At- and 125I-labeled closo-decaborate(2-) conjugates of Fab′ as a means of decreasing kidney retention

    Science.gov (United States)

    Wilbur, D. Scott; Chyan, Ming-Kuan; Hamlin, Donald K.; Nguyen, Holly; Vessella, Robert L.

    2011-01-01

    Evaluation of monoclonal antibody (MAb) fragments (e.g. Fab′, Fab or engineered fragments) as cancer-targeting reagents for therapy with the α-particle emitting radionuclide astatine-211 (211At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the 211At label, generally a meta- or para-[211At]astatobenzoyl conjugate, on MAb Fab′ fragments can be dramatically improved by use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled MAb Fab′ conjugates appears to result in retention of the radioactivity in kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of acid-cleavable hydrazone between the Fab′ and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete polyethylene glycol (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole or methyl) to provide varying acid-sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated (125I or 131I), then conjugated with an anti-PSMA Fab′ (107-1A4 Fab′). Biodistributions of the five radioiodinated Fab′ conjugates were obtained in nude mice at 1, 4 and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab′ through a non-cleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver and spleen that were similar to those obtained with directly radioiodinated Fab′ (i.e. no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate

  20. Domain walls collision in Fe-rich and Co-rich glass covered microwires

    Directory of Open Access Journals (Sweden)

    Gonzalez J.

    2013-01-01

    Full Text Available We report the results of the investigation of domain walls propagation in Fe-rich and Co-rich microwires performed using Sixtus-Tonks and magneto-optical Kerr effect techniques. It was found that under certain experimental conditions we are able to create the regime of the motion of two domain walls moving to opposite directions which terminates by the collision of the domain walls. Also the domain walls collision was visualized using magneto-optical Kerr effect microscope when the surface giant Barkhausen jump induced by circular magnetic field has been observed.

  1. Large-scale Identification of Expressed Sequence Tags (ESTs from Nicotianatabacum by Normalized cDNA Library Sequencing

    Directory of Open Access Journals (Sweden)

    Alvarez S Perez

    2014-12-01

    Full Text Available An expressed sequence tags (EST resource for tobacco plants (Nicotianatabacum was established using high-throughput sequencing of randomly selected clones from one cDNA library representing a range of plant organs (leaf, stem, root and root base. Over 5000 ESTs were generated from the 3’ ends of 8000 clones, analyzed by BLAST searches and categorized functionally. All annotated ESTs were classified into 18 functional categories, unique transcripts involved in energy were the largest group accounting for 831 (32.32% of the annotated ESTs. After excluding 2450 non-significant tentative unique transcripts (TUTs, 100 unique sequences (1.67% of total TUTs were identified from the N. tabacum database. In the array result two genes strongly related to the tobacco mosaic virus (TMV were obtained, one basic form of pathogenesis-related protein 1 precursor (TBT012G08 and ubiquitin (TBT087G01. Both of them were found in the variety Hongda, some other important genes were classified into two groups, one of these implicated in plant development like those genes related to a photosynthetic process (chlorophyll a-b binding protein, photosystem I, ferredoxin I and III, ATP synthase and a further group including genes related to plant stress response (ubiquitin, ubiquitin-like protein SMT3, glycine-rich RNA binding protein, histones and methallothionein. The interesting finding in this study is that two of these genes have never been reported before in N. tabacum (ubiquitin-like protein SMT3 and methallothionein. The array results were confirmed using quantitative PCR.

  2. TagDust2: a generic method to extract reads from sequencing data.

    Science.gov (United States)

    Lassmann, Timo

    2015-01-28

    Arguably the most basic step in the analysis of next generation sequencing data (NGS) involves the extraction of mappable reads from the raw reads produced by sequencing instruments. The presence of barcodes, adaptors and artifacts subject to sequencing errors makes this step non-trivial. Here I present TagDust2, a generic approach utilizing a library of hidden Markov models (HMM) to accurately extract reads from a wide array of possible read architectures. TagDust2 extracts more reads of higher quality compared to other approaches. Processing of multiplexed single, paired end and libraries containing unique molecular identifiers is fully supported. Two additional post processing steps are included to exclude known contaminants and filter out low complexity sequences. Finally, TagDust2 can automatically detect the library type of sequenced data from a predefined selection. Taken together TagDust2 is a feature rich, flexible and adaptive solution to go from raw to mappable NGS reads in a single step. The ability to recognize and record the contents of raw reads will help to automate and demystify the initial, and often poorly documented, steps in NGS data analysis pipelines. TagDust2 is freely available at: http://tagdust.sourceforge.net .

  3. Deciphering the biology of Mycobacterium tuberculosis from thecomplete genome sequence

    DEFF Research Database (Denmark)

    Cole, S.T.; Krogh, Anders Stærmose

    1998-01-01

    Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding....... tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation....

  4. Prostate Activated Prodrugs and Imaging Agents

    National Research Council Canada - National Science Library

    Jones, Graham B

    2004-01-01

    .... The substrate chosen was a 3 component system composed of a peptide sequence with affinity for PSA, an imaging agent and a deactivating bridge-linker, which electronically incapacitates the imaging agent...

  5. A GMBCG GALAXY CLUSTER CATALOG OF 55,424 RICH CLUSTERS FROM SDSS DR7

    International Nuclear Information System (INIS)

    Hao Jiangang; Annis, James; Johnston, David E.; McKay, Timothy A.; Evrard, August; Siegel, Seth R.; Gerdes, David; Koester, Benjamin P.; Rykoff, Eli S.; Rozo, Eduardo; Wechsler, Risa H.; Busha, Michael; Becker, Matthew; Sheldon, Erin

    2010-01-01

    We present a large catalog of optically selected galaxy clusters from the application of a new Gaussian Mixture Brightest Cluster Galaxy (GMBCG) algorithm to SDSS Data Release 7 data. The algorithm detects clusters by identifying the red-sequence plus brightest cluster galaxy (BCG) feature, which is unique for galaxy clusters and does not exist among field galaxies. Red-sequence clustering in color space is detected using an Error Corrected Gaussian Mixture Model. We run GMBCG on 8240 deg 2 of photometric data from SDSS DR7 to assemble the largest ever optical galaxy cluster catalog, consisting of over 55,000 rich clusters across the redshift range from 0.1 < z < 0.55. We present Monte Carlo tests of completeness and purity and perform cross-matching with X-ray clusters and with the maxBCG sample at low redshift. These tests indicate high completeness and purity across the full redshift range for clusters with 15 or more members.

  6. A GMBCG galaxy cluster catalog of 55,880 rich clusters from SDSS DR7

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Jiangang; McKay, Timothy A.; Koester, Benjamin P.; Rykoff, Eli S.; Rozo, Eduardo; Annis, James; Wechsler, Risa H.; Evrard, August; Siegel, Seth R.; Becker, Matthew; Busha, Michael; /Fermilab /Michigan U. /Chicago U., Astron. Astrophys. Ctr. /UC, Santa Barbara /KICP, Chicago /KIPAC, Menlo Park /SLAC /Caltech /Brookhaven

    2010-08-01

    We present a large catalog of optically selected galaxy clusters from the application of a new Gaussian Mixture Brightest Cluster Galaxy (GMBCG) algorithm to SDSS Data Release 7 data. The algorithm detects clusters by identifying the red sequence plus Brightest Cluster Galaxy (BCG) feature, which is unique for galaxy clusters and does not exist among field galaxies. Red sequence clustering in color space is detected using an Error Corrected Gaussian Mixture Model. We run GMBCG on 8240 square degrees of photometric data from SDSS DR7 to assemble the largest ever optical galaxy cluster catalog, consisting of over 55,000 rich clusters across the redshift range from 0.1 < z < 0.55. We present Monte Carlo tests of completeness and purity and perform cross-matching with X-ray clusters and with the maxBCG sample at low redshift. These tests indicate high completeness and purity across the full redshift range for clusters with 15 or more members.

  7. Structural elucidation, molecular representation and solvent interactions of vitrinite-rich and inertinite-rich South African coals

    Science.gov (United States)

    van Niekerk, Daniel

    The structural differences and similarities of two Permian-aged South African coals, vitrinite-rich Waterberg and inertinite-rich Highveld coals (similar rank, carbon content and Permian age), were evaluated. With South African coals the opportunity presented itself to study not only Permian-aged Gondwana vitrinite but also inertinite. It was expected that these coals would differ from Northern hemisphere Carboniferous coals. It was concluded from various structural data that both coals, although different in maceral composition and depositional basins, are similar in their base structural composition. The main differences were that the inertinite-rich Highveld coal was more ordered, more aromatic, and had less hydrogen than the vitrinite-rich Waterberg coal. Analytical data were used to construct large-scale advanced molecular representations for vitrinite-rich Waterberg and inertinite-rich Highveld coals. The three-dimensional models were structurally diverse with a molecular weight range of 78 to 1900 amu. The vitrinite-rich coal model consisted of 18,572 atoms and 191 individual molecules and the inertinite-rich coal model consisted of 14,242 atoms and 158 individual molecules. This largescale modeling effort was enabled by the development of various PERL scripts to automate various visualization and analytical aspects. Coal swelling studies were conducted using the traditional pack-bed swelling method and a new novel single-particle stop-motion videography swelling method with NMP and CS2/NMP solvents. The pack-bed swelling showed that vitrinite-rich coal had a greater swelling extent and that swelling extent for both coals was greater in CS2/NMP binary solvent than for NMP. Single-particle swelling experiments showed that both coals, for both solvents, exhibit overshoot-type and climbing-type swelling behaviors. Inertinite-coal had a faster swelling rate, in both solvents, than the vitrinite-rich coal. The single-particle swelling data was used to calculate

  8. Sequencing and annotation of mitochondrial genomes from individual parasitic helminths.

    Science.gov (United States)

    Jex, Aaron R; Littlewood, D Timothy; Gasser, Robin B

    2015-01-01

    Mitochondrial (mt) genomics has significant implications in a range of fundamental areas of parasitology, including evolution, systematics, and population genetics as well as explorations of mt biochemistry, physiology, and function. Mt genomes also provide a rich source of markers to aid molecular epidemiological and ecological studies of key parasites. However, there is still a paucity of information on mt genomes for many metazoan organisms, particularly parasitic helminths, which has often related to challenges linked to sequencing from tiny amounts of material. The advent of next-generation sequencing (NGS) technologies has paved the way for low cost, high-throughput mt genomic research, but there have been obstacles, particularly in relation to post-sequencing assembly and analyses of large datasets. In this chapter, we describe protocols for the efficient amplification and sequencing of mt genomes from small portions of individual helminths, and highlight the utility of NGS platforms to expedite mt genomics. In addition, we recommend approaches for manual or semi-automated bioinformatic annotation and analyses to overcome the bioinformatic "bottleneck" to research in this area. Taken together, these approaches have demonstrated applicability to a range of parasites and provide prospects for using complete mt genomic sequence datasets for large-scale molecular systematic and epidemiological studies. In addition, these methods have broader utility and might be readily adapted to a range of other medium-sized molecular regions (i.e., 10-100 kb), including large genomic operons, and other organellar (e.g., plastid) and viral genomes.

  9. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

  10. Linker-free 3D assembly of nanocrystals with tunable unit size for reversible lithium ion storage

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Da; Lee, Jim Yang, E-mail: cheleejy@nus.edu.sg [Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, 10 Kent Ridge Crescent, 119260 (Singapore)

    2011-09-02

    A simple and scalable procedure combining hydrothermal synthesis with post-synthesis calcination was developed to produce a linker-free, thermally stable, mesoscale 3D ordered assembly of spinel-type ZnCo{sub 2}O{sub 4} nanocrystals. The mesoscale assembly with distinctively sharp edges was formed by close-packing the ZnCo{sub 2}O{sub 4} nanocrystal building blocks with a unit size changeable by the synthesis temperature. A self-templating mechanism based on the topotactic transformation of an oxalato-bridged precursor coordination compound was proposed for the assembly. The packaging of crystalline ZnCo{sub 2}O{sub 4} nanoparticles, an active lithium ion storage compound, into a dense organized structure is an effective way to increase the volumetric capacity of ZnCo{sub 2}O{sub 4} nanoparticles for reversible lithium ion storage. The highly ordered 3D assembly of ZnCo{sub 2}O{sub 4} demonstrated excellent reversible lithium ion storage properties and a specific capacity ({approx}800 mAh g{sup -1}) much higher than that of carbon (typically {approx} 350 mAh g{sup -1}).

  11. Linker-free 3D assembly of nanocrystals with tunable unit size for reversible lithium ion storage

    International Nuclear Information System (INIS)

    Deng, Da; Lee, Jim Yang

    2011-01-01

    A simple and scalable procedure combining hydrothermal synthesis with post-synthesis calcination was developed to produce a linker-free, thermally stable, mesoscale 3D ordered assembly of spinel-type ZnCo 2 O 4 nanocrystals. The mesoscale assembly with distinctively sharp edges was formed by close-packing the ZnCo 2 O 4 nanocrystal building blocks with a unit size changeable by the synthesis temperature. A self-templating mechanism based on the topotactic transformation of an oxalato-bridged precursor coordination compound was proposed for the assembly. The packaging of crystalline ZnCo 2 O 4 nanoparticles, an active lithium ion storage compound, into a dense organized structure is an effective way to increase the volumetric capacity of ZnCo 2 O 4 nanoparticles for reversible lithium ion storage. The highly ordered 3D assembly of ZnCo 2 O 4 demonstrated excellent reversible lithium ion storage properties and a specific capacity (∼800 mAh g -1 ) much higher than that of carbon (typically ∼ 350 mAh g -1 ).

  12. Demonstration of Improved Charge Transfer in Graphene/Au Nanorods Plasmonic Hybrids Stabilized by Benzyl Thiol Linkers

    Directory of Open Access Journals (Sweden)

    Giuseppe Valerio Bianco

    2016-01-01

    Full Text Available Hybrids based on graphene decorated with plasmonic gold (Au nanostructures are being investigated as possible materials combination to add to graphene functionalities of tunable plasmon resonance and enhanced absorption at selected wavelength in the visible-near-infrared region of the spectrum. Here, we report a solution drop-casting approach for fabricating stable hybrids based on chemical vapor deposition (CVD graphene and Au nanorods, which are able to activate effective charge transfer from graphene. We demonstrate that CVD graphene functionalization by benzyl thiol (BZT provides the linker to strong anchoring, via S-Au bonds, Au nanorods to graphene. Optical measurements by spectroscopic ellipsometry give evidence of the introduction of plasmon resonances at 1.85 and 2.25 eV in the Au nanorods/BZT/graphene hybrids, which enable surface enhanced Raman scattering (SERS detection. Furthermore, an effective electron transfer from graphene to Au nanorods, resulting in an enhancement of p-type doping of graphene with a consequent decrease of its sheet resistance, is probed by Raman spectroscopy and corroborated by electrical measurements.

  13. Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors

    International Nuclear Information System (INIS)

    Chang, C.; Kokontis, J.; Liao, S.

    1988-01-01

    Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural Ars. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens

  14. Gold nanoparticles deposited on linker-free silicon substrate and embedded in aluminum Schottky contact.

    Science.gov (United States)

    Gorji, Mohammad Saleh; Razak, Khairunisak Abdul; Cheong, Kuan Yew

    2013-10-15

    Given the enormous importance of Au nanoparticles (NPs) deposition on Si substrates as the precursor for various applications, we present an alternative approach to deposit Au NPs on linker-free n- and p-type Si substrates. It is demonstrated that, all conditions being similar, there is a significant difference between densities of the deposited NPs on both substrates. The Zeta-potential and polarity of charges surrounding the hydroxylamine reduced seeded growth Au NPs, are determined by a Zetasizer. To investigate the surface properties of Si substrates, contact angle measurement is performed. Field-emission scanning electron microscope is then utilized to distinguish the NPs density on the substrates. Finally, Al/Si Schottky barrier diodes with embedded Au NPs are fabricated, and their structural and electrical characteristics are further evaluated using an energy-filtered transmission electron microscope and current-voltage measurements, respectively. The results reveal that the density of NPs is significantly higher on n-type Si substrate and consequently has more pronounced effects on the electrical characteristics of the diode. It is concluded that protonation of Si-OH group on Si surface in low pH is responsible for the immobilization of Au NPs, which eventually contributes to the lowering of barrier height and enhances the electrical characteristics. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. CBM RICH geometry optimization

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoud, Tariq; Hoehne, Claudia [II. Physikalisches Institut, Giessen Univ. (Germany); Collaboration: CBM-Collaboration

    2016-07-01

    The Compressed Baryonic Matter (CBM) experiment at the future FAIR complex will investigate the phase diagram of strongly interacting matter at high baryon density and moderate temperatures in A+A collisions from 2-11 AGeV (SIS100) beam energy. The main electron identification detector in the CBM experiment will be a RICH detector with a CO{sub 2} gaseous-radiator, focusing spherical glass mirrors, and MAPMT photo-detectors being placed on a PMT-plane. The RICH detector is located directly behind the CBM dipole magnet. As the final magnet geometry is now available, some changes in the RICH geometry become necessary. In order to guarantee a magnetic field of 1 mT at maximum in the PMT plane for effective operation of the MAPMTs, two measures have to be taken: The PMT plane is moved outwards of the stray field by tilting the mirrors by 10 degrees and shielding boxes have been designed. In this contribution the results of the geometry optimization procedure are presented.

  16. LITHIUM-RICH GIANTS IN GLOBULAR CLUSTERS

    Energy Technology Data Exchange (ETDEWEB)

    Kirby, Evan N.; Cohen, Judith G. [California Institute of Technology, 1200 E. California Boulevard, MC 249-17, Pasadena, CA 91125 (United States); Guhathakurta, Puragra [UCO/Lick Observatory and Department of Astronomy and Astrophysics, University of California, 1156 High Street, Santa Cruz, CA 95064 (United States); Zhang, Andrew J. [The Harker School, 500 Saratoga Avenue, San Jose, CA 95129 (United States); Hong, Jerry [Palo Alto High School, 50 Embarcadero Road, Palo Alto, CA, 94301 (United States); Guo, Michelle [Stanford University, 450 Serra Mall, Stanford, CA 94305 (United States); Guo, Rachel [Irvington High School, 41800 Blacow Road, Fremont, CA 94538 (United States); Cunha, Katia [Observatório Nacional, São Cristóvão Rio de Janeiro (Brazil)

    2016-03-10

    Although red giants deplete lithium on their surfaces, some giants are Li-rich. Intermediate-mass asymptotic giant branch (AGB) stars can generate Li through the Cameron–Fowler conveyor, but the existence of Li-rich, low-mass red giant branch (RGB) stars is puzzling. Globular clusters are the best sites to examine this phenomenon because it is straightforward to determine membership in the cluster and to identify the evolutionary state of each star. In 72 hours of Keck/DEIMOS exposures in 25 clusters, we found four Li-rich RGB and two Li-rich AGB stars. There were 1696 RGB and 125 AGB stars with measurements or upper limits consistent with normal abundances of Li. Hence, the frequency of Li-richness in globular clusters is (0.2 ± 0.1)% for the RGB, (1.6 ± 1.1)% for the AGB, and (0.3 ± 0.1)% for all giants. Because the Li-rich RGB stars are on the lower RGB, Li self-generation mechanisms proposed to occur at the luminosity function bump or He core flash cannot explain these four lower RGB stars. We propose the following origin for Li enrichment: (1) All luminous giants experience a brief phase of Li enrichment at the He core flash. (2) All post-RGB stars with binary companions on the lower RGB will engage in mass transfer. This scenario predicts that 0.1% of lower RGB stars will appear Li-rich due to mass transfer from a recently Li-enhanced companion. This frequency is at the lower end of our confidence interval.

  17. VisRseq: R-based visual framework for analysis of sequencing data.

    Science.gov (United States)

    Younesy, Hamid; Möller, Torsten; Lorincz, Matthew C; Karimi, Mohammad M; Jones, Steven J M

    2015-01-01

    Several tools have been developed to enable biologists to perform initial browsing and exploration of sequencing data. However the computational tool set for further analyses often requires significant computational expertise to use and many of the biologists with the knowledge needed to interpret these data must rely on programming experts. We present VisRseq, a framework for analysis of sequencing datasets that provides a computationally rich and accessible framework for integrative and interactive analyses without requiring programming expertise. We achieve this aim by providing R apps, which offer a semi-auto generated and unified graphical user interface for computational packages in R and repositories such as Bioconductor. To address the interactivity limitation inherent in R libraries, our framework includes several native apps that provide exploration and brushing operations as well as an integrated genome browser. The apps can be chained together to create more powerful analysis workflows. To validate the usability of VisRseq for analysis of sequencing data, we present two case studies performed by our collaborators and report their workflow and insights.

  18. Continuous Distributed Representation of Biological Sequences for Deep Proteomics and Genomics.

    Directory of Open Access Journals (Sweden)

    Ehsaneddin Asgari

    Full Text Available We introduce a new representation and feature extraction method for biological sequences. Named bio-vectors (BioVec to refer to biological sequences in general with protein-vectors (ProtVec for proteins (amino-acid sequences and gene-vectors (GeneVec for gene sequences, this representation can be widely used in applications of deep learning in proteomics and genomics. In the present paper, we focus on protein-vectors that can be utilized in a wide array of bioinformatics investigations such as family classification, protein visualization, structure prediction, disordered protein identification, and protein-protein interaction prediction. In this method, we adopt artificial neural network approaches and represent a protein sequence with a single dense n-dimensional vector. To evaluate this method, we apply it in classification of 324,018 protein sequences obtained from Swiss-Prot belonging to 7,027 protein families, where an average family classification accuracy of 93%±0.06% is obtained, outperforming existing family classification methods. In addition, we use ProtVec representation to predict disordered proteins from structured proteins. Two databases of disordered sequences are used: the DisProt database as well as a database featuring the disordered regions of nucleoporins rich with phenylalanine-glycine repeats (FG-Nups. Using support vector machine classifiers, FG-Nup sequences are distinguished from structured protein sequences found in Protein Data Bank (PDB with a 99.8% accuracy, and unstructured DisProt sequences are differentiated from structured DisProt sequences with 100.0% accuracy. These results indicate that by only providing sequence data for various proteins into this model, accurate information about protein structure can be determined. Importantly, this model needs to be trained only once and can then be applied to extract a comprehensive set of information regarding proteins of interest. Moreover, this representation can be

  19. Construction of porous cationic frameworks by crosslinking polyhedral oligomeric silsesquioxane units with N-heterocyclic linkers

    Science.gov (United States)

    Chen, Guojian; Zhou, Yu; Wang, Xiaochen; Li, Jing; Xue, Shuang; Liu, Yangqing; Wang, Qian; Wang, Jun

    2015-06-01

    In fields of materials science and chemistry, ionic-type porous materials attract increasing attention due to significant ion-exchanging capacity for accessing diversified applications. Facing the fact that porous cationic materials with robust and stable frameworks are very rare, novel tactics that can create new type members are highly desired. Here we report the first family of polyhedral oligomeric silsesquioxane (POSS) based porous cationic frameworks (PCIF-n) with enriched poly(ionic liquid)-like cationic structures, tunable mesoporosities, high surface areas (up to 1,025 m2 g-1) and large pore volumes (up to 0.90 cm3 g-1). Our strategy is designing the new rigid POSS unit of octakis(chloromethyl)silsesquioxane and reacting it with the rigid N-heterocyclic cross-linkers (typically 4,4‧-bipyridine) for preparing the desired porous cationic frameworks. The PCIF-n materials possess large surface area, hydrophobic and special anion-exchanging property, and thus are used as the supports for loading guest species PMo10V2O405- the resultant hybrid behaves as an efficient heterogeneous catalyst for aerobic oxidation of benzene and H2O2-mediated oxidation of cyclohexane.

  20. Replication error deficient and proficient colorectal cancer gene expression differences caused by 3'UTR polyT sequence deletions

    DEFF Research Database (Denmark)

    Wilding, Jennifer L; McGowan, Simon; Liu, Ying

    2010-01-01

    , and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, especially including message stability, are found in the 3'UTR sequence of most genes. The relevant sequences are typically A/U-rich elements or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER......- (proficient) colorectal cancer cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3'UTRs, 5'UTRs, and coding sequences of those genes most differentially expressed in RER+ versus RER- cell lines has shown that much...... of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3'UTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probesets for a large...

  1. Complete plastid genome sequence of Primula sinensis (Primulaceae: structure comparison, sequence variation and evidence for accD transfer to nucleus

    Directory of Open Access Journals (Sweden)

    Tong-Jian Liu

    2016-06-01

    Full Text Available Species-rich genus Primula L. is a typical plant group with which to understand genetic variance between species in different levels of relationships. Chloroplast genome sequences are used to be the information resource for quantifying this difference and reconstructing evolutionary history. In this study, we reported the complete chloroplast genome sequence of Primula sinensis and compared it with other related species. This genome of chloroplast showed a typical circular quadripartite structure with 150,859 bp in sequence length consisting of 37.2% GC base. Two inverted repeated regions (25,535 bp were separated by a large single-copy region (82,064 bp and a small single-copy region (17,725 bp. The genome consists of 112 genes, including 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Among them, seven coding genes, seven tRNA genes and four rRNA genes have two copies due to their locations in the IR regions. The accD and infA genes lacking intact open reading frames (ORF were identified as pseudogenes. SSR and sequence variation analyses were also performed on the plastome of Primula sinensis, comparing with another available plastome of P. poissonii. The four most variable regions, rpl36–rps8, rps16–trnQ, trnH–psbA and ndhC–trnV, were identified. Phylogenetic relationship estimates using three sub-datasets extracted from a matrix of 57 protein-coding gene sequences showed the identical result that was consistent with previous studies. A transcript found from P. sinensis transcriptome showed a high similarity to plastid accD functional region and was identified as a putative plastid transit peptide at the N-terminal region. The result strongly suggested that plastid accD has been functionally transferred to the nucleus in P. sinensis.

  2. Complete Genome Sequence of Streptomyces albus SM254, a Potent Antagonist of Bat White-Nose Syndrome Pathogen Pseudogymnoascus destructans.

    Science.gov (United States)

    Badalamenti, Jonathan P; Erickson, Joshua D; Salomon, Christine E

    2016-04-14

    We sequenced and annotated the complete 7,170,504-bp genome of a novel secondary metabolite-producingStreptomycesstrain,Streptomyces albusSM254, isolated from copper-rich subsurface fluids at ~220-m depth within the Soudan Iron Mine (Soudan, MN, USA). Copyright © 2016 Badalamenti et al.

  3. Yedomas in Alaska: Evolution of ice-rich landscapes in a changing climate

    Science.gov (United States)

    Stephani, E.; Kanevskiy, M. Z.; Fortier, D.; Shur, Y.; Jorgenson, T. T.; Dillon, M.; Bray, M.

    2011-12-01

    Yedomas (Ice complexes) have developed on lands that remained unglaciated during the Late-Pleistocene. Ground exposure to cold climate allowed large syngenetic ice wedges to form typically in fine-grained, organic-rich, and ice-rich enclosing sediments, resulting in particularly ice-rich and thick sequences. Changing climate since has triggered geomorphological changes of these ice-rich landscapes and now contemporary climate conditions generally favour their degradation. Yedoma remnants have been observed in areas of Alaska including in the northern part of Seward Peninsula and Iktilik River area where we studied their metrics, cryostratigraphy, soil properties, and their degradation processes. Understanding the dynamic of this particular periglacial landscape and determining its properties is essential for modeling its future evolution in a changing climate. At our three study sites, presence of typical geomorphological features and cryostratigraphic units revealed information on the landscape evolution since deposition of these ice-rich strata. A Yedoma deposit in the northern part of Seward Peninsula comprised ice wedges at least 36 m-deep. The enclosing sediment was characterized by an ice-rich cryofacies of coarse silt with microlenticular cryostructure and abundant fine rootlets. The intermediate layer, a typical extremely ice-rich layer located below the active layer, was observed above the Yedoma deposit in areas less affected by thermo-degradation. In the thermo-degraded areas characterized by an irregular terrain surface, the intermediate layer was replaced by the generally ice-poor taberal cryofacies which corresponds to a deposit that was formerly ice-rich, thawed, drained, and eventually refrozen. Yedoma remnants in their contemporary degrading state can be recognized with their abundant thermokarst lakes, drained lake basins, and drainage gullies. Thermokarst lakes can be particularly deep because of the considerable amount of ground ice that can

  4. Mixed-linker UiO-66: structure-property relationships revealed by a combination of high-resolution powder X-ray diffraction and density functional theory calculations.

    Science.gov (United States)

    Taddei, Marco; Tiana, Davide; Casati, Nicola; van Bokhoven, Jeroen A; Smit, Berend; Ranocchiari, Marco

    2017-01-04

    The use of mixed-linker metal-organic frameworks (MIXMOFs) is one of the most effective strategies to modulate the physical-chemical properties of MOFs without affecting the overall crystal structure. In many instances, MIXMOFs have been recognized as solid solutions, with random distribution of ligands, in agreement with the empirical rule known as Vegard's law. In this work, we have undertaken a study combining high-resolution powder X-ray diffraction (HR-PXRD) and density functional theory (DFT) calculations with the aim of understanding the reasons why UiO-66-based amino- and bromo-functionalized MIXMOFs (MIXUiO-66) undergo cell expansion obeying Vegard's law and how this behaviour is related to their physical-chemical properties. DFT calculations predict that the unit cell in amino-functionalized UiO-66 experiences only minor expansion as a result of steric effects, whereas major modification to the electronic features of the framework leads to weaker metal-linker interaction and consequently to the loss of stability at higher degrees of functionalization. For bromo-functionalized UiO-66, steric repulsion due to the size of bromine yields a large cell expansion, but the electronic features remain very similar to pristine UiO-66, preserving the stability of the framework upon functionalization. MIXUiO-66 obtained by either direct synthesis or by post-synthetic exchange shows Vegard-like behaviour, suggesting that both preparation methods yield solid solutions, but the thermal stability and the textural properties of the post-synthetic exchanged materials do not display a clear dependence on the chemical composition, as observed for the MOFs obtained by direct synthesis.

  5. Application of a continuously stirred tank bioreactor (CSTR) for bioremediation of hydrocarbon-rich industrial wastewater effluents.

    Science.gov (United States)

    Gargouri, Boutheina; Karray, Fatma; Mhiri, Najla; Aloui, Fathi; Sayadi, Sami

    2011-05-15

    A continuously stirred tank bioreactor (CSTR) was used to optimize feasible and reliable bioprocess system in order to treat hydrocarbon-rich industrial wastewaters. A successful bioremediation was developed by an efficient acclimatized microbial consortium. After an experimental period of 225 days, the process was shown to be highly efficient in decontaminating the wastewater. The performance of the bioaugmented reactor was demonstrated by the reduction of COD rates up to 95%. The residual total petroleum hydrocarbon (TPH) decreased from 320 mg TPH l(-1) to 8 mg TPH l(-1). Analysis using gas chromatography-mass spectrometry (GC-MS) identified 26 hydrocarbons. The use of the mixed cultures demonstrated high degradation performance for hydrocarbons range n-alkanes (C10-C35). Six microbial isolates from the CSTR were characterized and species identification was confirmed by sequencing the 16S rRNA genes. The partial 16S rRNA gene sequences demonstrated that 5 strains were closely related to Aeromonas punctata (Aeromonas caviae), Bacillus cereus, Ochrobactrum intermedium, Stenotrophomonas maltophilia and Rhodococcus sp. The 6th isolate was affiliated to genera Achromobacter. Besides, the treated wastewater could be considered as non toxic according to the phytotoxicity test since the germination index of Lepidium sativum ranged between 57 and 95%. The treatment provided satisfactory results and presents a feasible technology for the treatment of hydrocarbon-rich wastewater from petrochemical industries and petroleum refineries. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Detection of G-Quadruplex Structures Formed by G-Rich Sequences from Rice Genome and Transcriptome Using Combined Probes.

    Science.gov (United States)

    Chang, Tianjun; Li, Weiguo; Ding, Zhan; Cheng, Shaofei; Liang, Kun; Liu, Xiangjun; Bing, Tao; Shangguan, Dihua

    2017-08-01

    Putative G-quadruplex (G4) forming sequences (PQS) are highly prevalent in the genome and transcriptome of various organisms and are considered as potential regulation elements in many biological processes by forming G4 structures. The formation of G4 structures highly depends on the sequences and the environment. In most cases, it is difficult to predict G4 formation by PQS, especially PQS containing G2 tracts. Therefore, the experimental identification of G4 formation is essential in the study of G4-related biological functions. Herein, we report a rapid and simple method for the detection of G4 structures by using a pair of complementary reporters, hemin and BMSP. This method was applied to detect G4 structures formed by PQS (DNA and RNA) searched in the genome and transcriptome of Oryza sativa. Unlike most of the reported G4 probes that only recognize part of G4 structures, the proposed method based on combined probes positively responded to almost all G4 conformations, including parallel, antiparallel, and mixed/hybrid G4, but did not respond to non-G4 sequences. This method shows potential for high-throughput identification of G4 structures in genome and transcriptome. Furthermore, BMSP was observed to drive some PQS to form more stable G4 structures or induce the G4 formation of some PQS that cannot form G4 in normal physiological conditions, which may provide a powerful molecular tool for gene regulation.

  7. Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

    Science.gov (United States)

    Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

    2017-11-01

    We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

  8. A global view of structure-function relationships in the tautomerase superfamily.

    Science.gov (United States)

    Davidson, Rebecca; Baas, Bert-Jan; Akiva, Eyal; Holliday, Gemma L; Polacco, Benjamin J; LeVieux, Jake A; Pullara, Collin R; Zhang, Yan Jessie; Whitman, Christian P; Babbitt, Patricia C

    2018-02-16

    The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure-function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis -3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase-like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Complete mitochondrial genome sequence of the hedgehog seahorse Hippocampus spinosissimus Weber, 1933 (Gasterosteiformes:Syngnathidae).

    Science.gov (United States)

    Liu, Shuaishuai; Zhang, Yanhong; Wang, Changming; Lin, Qiang

    2016-07-01

    The complete mitochondrial genome sequence of the hedgehog seahorse Hippocampus spinosissimus was first determined in this article. The total length of H. spinosissimus mitogenome is 16 527 bp and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region. The gene order and composition of H. spinosissimus were similar to those of most other vertebrates. The overall base composition of H. spinosissimus is 32.1% A, 30.3% T, 14.9% G and 22.7% C, with a slight A + T-rich feature (62.4%). Phylogenetic analyses based on complete mitochondrial genome sequence showed that H. spinosissimus has a close genetic relationship to H. ingens and H. kuda.

  10. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

    DEFF Research Database (Denmark)

    Guðnason, Haukur; Dufva, Hans Martin; Bang, Dang Duong

    2007-01-01

    investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit......The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include...... the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Although a few alternative dyes without some of these limitations have been recently proposed, no large-scale investigation into the properties of intercalating dyes has been performed. In this study, we...

  11. A facile cathode design combining Ni-rich layered oxides with Li-rich layered oxides for lithium-ion batteries

    Energy Technology Data Exchange (ETDEWEB)

    Song, Bohang; Li, Wangda; Yan, Pengfei; Oh, Seung-Min; Wang, Chong-Min; Manthiram, Arumugam

    2016-09-01

    A facile synthesis method was developed to prepare xLi2MnO3·(1-x)LiNi0.7Co0.15Mn0.15O2 (x = 0, 0.03, 0.07, 0.10, 0.20, and 0.30 as molar ratio) cathode materials, combining the advantages of high specific capacity from Ni-rich layered phase and surface chemical stability from Li-rich layered phase. X-ray diffraction (XRD), high resolution transmission electron microscope (HRTEM) and electrochemical charge/discharge performance confirm the formation of a Li-rich layered phase with C2/m symmetry. Most importantly, high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) reveals a spatial relationship that Li-rich nano-domain islands are integrated into a conventional Ni-rich layered matrix (R$\\bar{3}$m). This is the first time that Li-rich phase has been directly observed inside a particle at the nano-scale, when the overall composition of layered compounds (Li1+δNixMnyM1-x-y-δO2, M refers to transition metal elements) is Ni-rich (x > 0.5) rather than Mn-rich (y > 0.5). Remarkably, xLi2MnO3·(1-x)LiNi0.7Co0.15Mn0.15O2 cathode with optimized x value shows superior electrochemical performance at C/3, i.e., 170 mA h g-1 with 90.3 % of capacity retention after 400 cycles at 25 °C and 164 mA h g-1 with 81.3 % capacity retention after 200 cycles at 55 °C.

  12. Sequence, 'subtle' alternative splicing and expression of the CYYR1 (cysteine/tyrosine-rich 1) mRNA in human neuroendocrine tumors

    International Nuclear Information System (INIS)

    Vitale, Lorenza; Coppola, Domenico; Strippoli, Pierluigi; Frabetti, Flavia; Huntsman, Shane A; Canaider, Silvia; Casadei, Raffaella; Lenzi, Luca; Facchin, Federica; Carinci, Paolo; Zannotti, Maria

    2007-01-01

    CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG - isoform, the first described mRNA for CYYR1 locus) or the presence (CAG + isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG - or CAG + isoform expression compared to control tissues. CYYR1 CAG - isoform was significantly more expressed than CAG + isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. A new 'subtle' splicing isoform (CAG + ) of CYYR1 mRNA, the sequence and

  13. Sequence spaces [Formula: see text] and [Formula: see text] with application in clustering.

    Science.gov (United States)

    Khan, Mohd Shoaib; Alamri, Badriah As; Mursaleen, M; Lohani, Qm Danish

    2017-01-01

    Distance measures play a central role in evolving the clustering technique. Due to the rich mathematical background and natural implementation of [Formula: see text] distance measures, researchers were motivated to use them in almost every clustering process. Beside [Formula: see text] distance measures, there exist several distance measures. Sargent introduced a special type of distance measures [Formula: see text] and [Formula: see text] which is closely related to [Formula: see text]. In this paper, we generalized the Sargent sequence spaces through introduction of [Formula: see text] and [Formula: see text] sequence spaces. Moreover, it is shown that both spaces are BK -spaces, and one is a dual of another. Further, we have clustered the two-moon dataset by using an induced [Formula: see text]-distance measure (induced by the Sargent sequence space [Formula: see text]) in the k-means clustering algorithm. The clustering result established the efficacy of replacing the Euclidean distance measure by the [Formula: see text]-distance measure in the k-means algorithm.

  14. Improvement of methods for large scale sequencing; application to human Xq28

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, R.A.; Andersson, B.; Wentland, M.A. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Sequencing of a one-metabase region of Xq28, spanning the FRAXA and IDS loci has been undertaken in order to investigate the practicality of the shotgun approach for large scale sequencing and as a platform to develop improved methods. The efficiency of several steps in the shotgun sequencing strategy has been increased using PCR-based approaches. An improved method for preparation of M13 libraries has been developed. This protocol combines a previously described adaptor-based protocol with the uracil DNA glycosylase (UDG)-cloning procedure. The efficiency of this procedure has been found to be up to 100-fold higher than that of previously used protocols. In addition the novel protocol is more reliable and thus easy to establish in a laboratory. The method has also been adapted for the simultaneous shotgun sequencing of multiple short fragments by concentrating them before library construction is presented. This protocol is suitable for rapid characterization of cDNA clones. A library was constructed from 15 PCR-amplified and concentrated human cDNA inserts, and the insert sequences could easily be identified as separate contigs during the assembly process and the sequence coverage was even along each fragment. Using this strategy, the fine structures of the FraxA and IDS loci have been revealed and several EST homologies indicating novel expressed sequences have been identified. Use of PCR to close repetitive regions that are difficult to clone was tested by determination of the sequence of a cosmid mapping DXS455 in Xq28, containing a polymorphic VNTR. The region containing the VNTR was not represented in the shotgun library, but by designing PCR primers in the sequences flanking the gap and by cloning and sequencing the PCR product, the fine structure of the VNTR has been determined. It was found to be an AT-rich VNTR with a repeated 25-mer at the center.

  15. Sequence tagging reveals unexpected modifications in toxicoproteomics

    Science.gov (United States)

    Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

    2010-01-01

    Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications. PMID:21214251

  16. NEW COOLING SEQUENCES FOR OLD WHITE DWARFS

    International Nuclear Information System (INIS)

    Renedo, I.; Althaus, L. G.; GarcIa-Berro, E.; Miller Bertolami, M. M.; Romero, A. D.; Corsico, A. H.; Rohrmann, R. D.

    2010-01-01

    We present full evolutionary calculations appropriate for the study of hydrogen-rich DA white dwarfs. This is done by evolving white dwarf progenitors from the zero-age main sequence, through the core hydrogen-burning phase, the helium-burning phase, and the thermally pulsing asymptotic giant branch phase to the white dwarf stage. Complete evolutionary sequences are computed for a wide range of stellar masses and for two different metallicities, Z = 0.01, which is representative of the solar neighborhood, and Z = 0.001, which is appropriate for the study of old stellar systems, like globular clusters. During the white dwarf cooling stage, we self-consistently compute the phase in which nuclear reactions are still important, the diffusive evolution of the elements in the outer layers and, finally, we also take into account all the relevant energy sources in the deep interior of the white dwarf, such as the release of latent heat and the release of gravitational energy due to carbon-oxygen phase separation upon crystallization. We also provide colors and magnitudes for these sequences, based on a new set of improved non-gray white dwarf model atmospheres, which include the most up-to-date physical inputs like the Lyα quasi-molecular opacity. The calculations are extended down to an effective temperature of 2500 K. Our calculations provide a homogeneous set of evolutionary cooling tracks appropriate for mass and age determinations of old DA white dwarfs and for white dwarf cosmochronology of the different Galactic populations.

  17. Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

    Science.gov (United States)

    Schnall-Levin, Michael; Rissland, Olivia S.; Johnston, Wendy K.; Perrimon, Norbert; Bartel, David P.; Berger, Bonnie

    2011-01-01

    MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. PMID:21685129

  18. Platelet-rich fibrin: the benefits.

    Science.gov (United States)

    Kumar, Yuvika Raj; Mohanty, Sujata; Verma, Mahesh; Kaur, Raunaq Reet; Bhatia, Priyanka; Kumar, Varun Raj; Chaudhary, Zainab

    2016-01-01

    Current published data presents confusing results about the effects of platelet-rich fibrin on bone, and there is a need for studies that throw light on its effect. Our main objective therefore was to evaluate (by fractal analysis) osseous regeneration in extraction sockets with and without platelet-rich fibrin in a study with a substantial sample and a reliable technique to calibrate its effects on bone cells. We also assessed the soft tissue response. Thirty-four patients had their bilaterally impacted third molars (68 surgical sites) extracted in this split-mouth study, following which platelet-rich fibrin was placed in one of the sockets. Patients were followed up clinically and radiographically, and a pain score and fractal analysis were used to evaluate healing of soft tissue and bone, respectively. We conclude that platelet-rich fibrin improves healing of both soft and hard tissues. Although osseous healing did not differ significantly between the groups, healing of soft tissue as judged by the pain score was significantly better in the experimental group. Copyright © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  19. Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

    Science.gov (United States)

    Liu, X; Gorovsky, M A

    1996-01-01

    A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced. PMID:8760889

  20. PRECISION MEASUREMENTS OF THE CLUSTER RED SEQUENCE USING AN ERROR-CORRECTED GAUSSIAN MIXTURE MODEL

    International Nuclear Information System (INIS)

    Hao Jiangang; Annis, James; Koester, Benjamin P.; Mckay, Timothy A.; Evrard, August; Gerdes, David; Rykoff, Eli S.; Rozo, Eduardo; Becker, Matthew; Busha, Michael; Wechsler, Risa H.; Johnston, David E.; Sheldon, Erin

    2009-01-01

    The red sequence is an important feature of galaxy clusters and plays a crucial role in optical cluster detection. Measurement of the slope and scatter of the red sequence are affected both by selection of red sequence galaxies and measurement errors. In this paper, we describe a new error-corrected Gaussian Mixture Model for red sequence galaxy identification. Using this technique, we can remove the effects of measurement error and extract unbiased information about the intrinsic properties of the red sequence. We use this method to select red sequence galaxies in each of the 13,823 clusters in the maxBCG catalog, and measure the red sequence ridgeline location and scatter of each. These measurements provide precise constraints on the variation of the average red galaxy populations in the observed frame with redshift. We find that the scatter of the red sequence ridgeline increases mildly with redshift, and that the slope decreases with redshift. We also observe that the slope does not strongly depend on cluster richness. Using similar methods, we show that this behavior is mirrored in a spectroscopic sample of field galaxies, further emphasizing that ridgeline properties are independent of environment. These precise measurements serve as an important observational check on simulations and mock galaxy catalogs. The observed trends in the slope and scatter of the red sequence ridgeline with redshift are clues to possible intrinsic evolution of the cluster red sequence itself. Most importantly, the methods presented in this work lay the groundwork for further improvements in optically based cluster cosmology.

  1. Precision Measurements of the Cluster Red Sequence using an Error Corrected Gaussian Mixture Model

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Jiangang; /Fermilab /Michigan U.; Koester, Benjamin P.; /Chicago U.; Mckay, Timothy A.; /Michigan U.; Rykoff, Eli S.; /UC, Santa Barbara; Rozo, Eduardo; /Ohio State U.; Evrard, August; /Michigan U.; Annis, James; /Fermilab; Becker, Matthew; /Chicago U.; Busha, Michael; /KIPAC, Menlo Park /SLAC; Gerdes, David; /Michigan U.; Johnston, David E.; /Northwestern U. /Brookhaven

    2009-07-01

    The red sequence is an important feature of galaxy clusters and plays a crucial role in optical cluster detection. Measurement of the slope and scatter of the red sequence are affected both by selection of red sequence galaxies and measurement errors. In this paper, we describe a new error corrected Gaussian Mixture Model for red sequence galaxy identification. Using this technique, we can remove the effects of measurement error and extract unbiased information about the intrinsic properties of the red sequence. We use this method to select red sequence galaxies in each of the 13,823 clusters in the maxBCG catalog, and measure the red sequence ridgeline location and scatter of each. These measurements provide precise constraints on the variation of the average red galaxy populations in the observed frame with redshift. We find that the scatter of the red sequence ridgeline increases mildly with redshift, and that the slope decreases with redshift. We also observe that the slope does not strongly depend on cluster richness. Using similar methods, we show that this behavior is mirrored in a spectroscopic sample of field galaxies, further emphasizing that ridgeline properties are independent of environment. These precise measurements serve as an important observational check on simulations and mock galaxy catalogs. The observed trends in the slope and scatter of the red sequence ridgeline with redshift are clues to possible intrinsic evolution of the cluster red-sequence itself. Most importantly, the methods presented in this work lay the groundwork for further improvements in optically-based cluster cosmology.

  2. Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

    Directory of Open Access Journals (Sweden)

    Artyom A Alekseyenko

    Full Text Available The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE. However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex and female Kc cells (which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

  3. A Novel Membrane-Permeable Breast-Targeting Pro-Apoptotic Peptide for Treatment of Breast Cancer

    National Research Council Canada - National Science Library

    Guo, Bin

    2005-01-01

    .... The BH3 peptide of Bid (EDIIRNIARHLAQVGDSMDR) was synthesized with eight d-arginine residues at the N-terminus with a glycine linker residue, followed by a breast-homing sequence (CPGPEGAGC) at the C-terminal...

  4. Platelet-Rich Plasma Increases Pigmentation.

    Science.gov (United States)

    Uysal, Cagri A; Ertas, Nilgun Markal

    2017-11-01

    Platelet-rich plasma (PRP) is an autologous solution of plasma containing 4 to 7 times the baseline concentration of human platelets. Platelet-rich plasma has been widely popular in facial rejuvenation to attenuate wrinkles and has been practically used. The authors have been encountering various patients of increased hiperpigmentation following PRP applications that were performed to attenuate the postinflammatory hiperpigmentation especially after laser treatment. The authors have been using PRP for facial rejuvenation in selected patients and in 1 patient the authors have encountered increased pigmentation over the pigmented skin lesions that were present before the application. The authors recommend that the PRP might increase pigmentation especially in the face region and precautions might be taken before and after the application. Platelet-rich plasma should not be used for the treatment of post inflammatory hiperpigmentation.

  5. Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes.

    Science.gov (United States)

    Johnson, Kim L; Cassin, Andrew M; Lonsdale, Andrew; Wong, Gane Ka-Shu; Soltis, Douglas E; Miles, Nicholas W; Melkonian, Michael; Melkonian, Barbara; Deyholos, Michael K; Leebens-Mack, James; Rothfels, Carl J; Stevenson, Dennis W; Graham, Sean W; Wang, Xumin; Wu, Shuangxiu; Pires, J Chris; Edger, Patrick P; Carpenter, Eric J; Bacic, Antony; Doblin, Monika S; Schultz, Carolyn J

    2017-06-01

    The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SP n EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots. © 2017 American Society of Plant Biologists. All Rights Reserved.

  6. Ag2S Quantum Dot-Sensitized Solar Cells by First Principles: The Effect of Capping Ligands and Linkers.

    Science.gov (United States)

    Amaya Suárez, Javier; Plata, Jose J; Márquez, Antonio M; Fernández Sanz, Javier

    2017-09-28

    Quantum dots solar cells, QDSCs, are one of the candidates for being a reliable alternative to fossil fuels. However, the well-studied CdSe and CdTe-based QDSCs present a variety of issues for their use in consumer-goods applications. Silver sulfide, Ag 2 S, is a promising material, but poor efficiency has been reported for QDSCs based on this compound. The potential influence of each component of QDSCs is critical and key for the development of more efficient devices based on Ag 2 S. In this work, density functional theory calculations were performed to study the nature of the optoelectronic properties for an anatase-TiO 2 (101) surface sensitized with different silver sulfide nanoclusters. We demonstrated how it is possible to deeply tune of its electronic properties by modifying the capping ligands and linkers to the surface. Finally, an analysis of the electron injection mechanism for this system is presented.

  7. Organic farming increases richness of fungal taxa in the wheat phyllosphere.

    Science.gov (United States)

    Karlsson, Ida; Friberg, Hanna; Kolseth, Anna-Karin; Steinberg, Christian; Persson, Paula

    2017-07-01

    Organic farming is often advocated as an approach to mitigate biodiversity loss on agricultural land. The phyllosphere provides a habitat for diverse fungal communities that are important for plant health and productivity. However, it is still unknown how organic farming affects the diversity of phyllosphere fungi in major crops. We sampled wheat leaves from 22 organically and conventionally cultivated fields in Sweden, paired based on their geographical location and wheat cultivar. Fungal communities were described using amplicon sequencing and real-time PCR. Species richness was higher on wheat leaves from organically managed fields, with a mean of 54 operational taxonomic units (OTUs) compared with 40 OTUs for conventionally managed fields. The main components of the fungal community were similar throughout the 350-km-long sampling area, and seven OTUs were present in all fields: Zymoseptoria, Dioszegia fristingensis, Cladosporium, Dioszegia hungarica, Cryptococcus, Ascochyta and Dioszegia. Fungal abundance was highly variable between fields, 10 3 -10 5 internal transcribed spacer copies per ng wheat DNA, but did not differ between cropping systems. Further analyses showed that weed biomass was the strongest explanatory variable for fungal community composition and OTU richness. These findings help provide a more comprehensive understanding of the effect of organic farming on the diversity of organism groups in different habitats within the agroecosystem. © 2017 The Authors Molecular Ecology Published by John Wiley & Sons Ltd.

  8. Quantitative analysis and prediction of curvature in leucine-rich repeat proteins.

    Science.gov (United States)

    Hindle, K Lauren; Bella, Jordi; Lovell, Simon C

    2009-11-01

    Leucine-rich repeat (LRR) proteins form a large and diverse family. They have a wide range of functions most of which involve the formation of protein-protein interactions. All known LRR structures form curved solenoids, although there is large variation in their curvature. It is this curvature that determines the shape and dimensions of the inner space available for ligand binding. Unfortunately, large-scale parameters such as the overall curvature of a protein domain are extremely difficult to predict. Here, we present a quantitative analysis of determinants of curvature of this family. Individual repeats typically range in length between 20 and 30 residues and have a variety of secondary structures on their convex side. The observed curvature of the LRR domains correlates poorly with the lengths of their individual repeats. We have, therefore, developed a scoring function based on the secondary structure of the convex side of the protein that allows prediction of the overall curvature with a high degree of accuracy. We also demonstrate the effectiveness of this method in selecting a suitable template for comparative modeling. We have developed an automated, quantitative protocol that can be used to predict accurately the curvature of leucine-rich repeat proteins of unknown structure from sequence alone. This protocol is available as an online resource at http://www.bioinf.manchester.ac.uk/curlrr/.

  9. The CBM RICH project

    Energy Technology Data Exchange (ETDEWEB)

    Adamczewski-Musch, J. [GSI Darmstadt (Germany); Becker, K.-H. [University Wuppertal (Germany); Belogurov, S. [ITEP Moscow (Russian Federation); Boldyreva, N. [PNPI Gatchina (Russian Federation); Chernogorov, A. [ITEP Moscow (Russian Federation); Deveaux, C. [University Gießen (Germany); Dobyrn, V. [PNPI Gatchina (Russian Federation); Dürr, M. [University Gießen (Germany); Eom, J. [Pusan National University (Korea, Republic of); Eschke, J. [GSI Darmstadt (Germany); Höhne, C. [University Gießen (Germany); Kampert, K.-H. [University Wuppertal (Germany); Kleipa, V. [GSI Darmstadt (Germany); Kochenda, L. [PNPI Gatchina (Russian Federation); Kolb, B. [GSI Darmstadt (Germany); Kopfer, J. [University Wuppertal (Germany); Kravtsov, P. [PNPI Gatchina (Russian Federation); Lebedev, S.; Lebedeva, E. [University Gießen (Germany); Leonova, E. [PNPI Gatchina (Russian Federation); and others

    2014-12-01

    The Compressed Baryonic Matter (CBM) experiment will study the properties of super dense nuclear matter by means of heavy ion collisions at the future FAIR facility. An integral detector component is a large Ring Imaging Cherenkov detector with CO{sub 2} gas radiator, which will mainly serve for electron identification and pion suppression necessary to access rare dileptonic probes like e{sup +}e{sup −} decays of light vector mesons or J/Ψ. We describe the design of this future RICH detector and focus on results obtained by building a CBM RICH detector prototype tested at CERN-PS.

  10. Scoring protein relationships in functional interaction networks predicted from sequence data.

    Directory of Open Access Journals (Sweden)

    Gaston K Mazandu

    Full Text Available UNLABELLED: The abundance of diverse biological data from various sources constitutes a rich source of knowledge, which has the power to advance our understanding of organisms. This requires computational methods in order to integrate and exploit these data effectively and elucidate local and genome wide functional connections between protein pairs, thus enabling functional inferences for uncharacterized proteins. These biological data are primarily in the form of sequences, which determine functions, although functional properties of a protein can often be predicted from just the domains it contains. Thus, protein sequences and domains can be used to predict protein pair-wise functional relationships, and thus contribute to the function prediction process of uncharacterized proteins in order to ensure that knowledge is gained from sequencing efforts. In this work, we introduce information-theoretic based approaches to score protein-protein functional interaction pairs predicted from protein sequence similarity and conserved protein signature matches. The proposed schemes are effective for data-driven scoring of connections between protein pairs. We applied these schemes to the Mycobacterium tuberculosis proteome to produce a homology-based functional network of the organism with a high confidence and coverage. We use the network for predicting functions of uncharacterised proteins. AVAILABILITY: Protein pair-wise functional relationship scores for Mycobacterium tuberculosis strain CDC1551 sequence data and python scripts to compute these scores are available at http://web.cbio.uct.ac.za/~gmazandu/scoringschemes.

  11. Complete chloroplast genome sequence of a major economic species, Ziziphus jujuba (Rhamnaceae).

    Science.gov (United States)

    Ma, Qiuyue; Li, Shuxian; Bi, Changwei; Hao, Zhaodong; Sun, Congrui; Ye, Ning

    2017-02-01

    Ziziphus jujuba is an important woody plant with high economic and medicinal value. Here, we analyzed and characterized the complete chloroplast (cp) genome of Z. jujuba, the first member of the Rhamnaceae family for which the chloroplast genome sequence has been reported. We also built a web browser for navigating the cp genome of Z. jujuba ( http://bio.njfu.edu.cn/gb2/gbrowse/Ziziphus_jujuba_cp/ ). Sequence analysis showed that this cp genome is 161,466 bp long and has a typical quadripartite structure of large (LSC, 89,120 bp) and small (SSC, 19,348 bp) single-copy regions separated by a pair of inverted repeats (IRs, 26,499 bp). The sequence contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The genome structure, gene order, GC content, and codon usage are similar to other typical angiosperm cp genomes. A total of 38 tandem repeats, two forward repeats, and three palindromic repeats were detected in the Z. jujuba cp genome. Simple sequence repeat (SSR) analysis revealed that most SSRs were AT-rich. The homopolymer regions in the cp genome of Z. jujuba were verified and manually corrected by Sanger sequencing. One-third of mononucleotide repeats were found to be erroneously sequenced by the 454 pyrosequencing, which resulted in sequences of 1-4 bases shorter than that by the Sanger sequencing. Analyzing the cp genome of Z. jujuba revealed that the IR contraction and expansion events resulted in ycf1 and rps19 pseudogenes. A phylogenetic analysis based on 64 protein-coding genes showed that Z. jujuba was closely related to members of the Elaeagnaceae family, which will be helpful for phylogenetic studies of other Rosales species. The complete cp genome sequence of Z. jujuba will facilitate population, phylogenetic, and cp genetic engineering studies of this economic plant.

  12. A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

    Science.gov (United States)

    Tan, Swee Jin; Phan, Huan; Gerry, Benjamin Michael; Kuhn, Alexandre; Hong, Lewis Zuocheng; Min Ong, Yao; Poon, Polly Suk Yean; Unger, Marc Alexander; Jones, Robert C.; Quake, Stephen R.; Burkholder, William F.

    2013-01-01

    Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation. PMID:23894273

  13. A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

    Directory of Open Access Journals (Sweden)

    Swee Jin Tan

    Full Text Available Library preparation for next-generation DNA sequencing (NGS remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

  14. An Automatic Matcher and Linker for Transportation Datasets

    Directory of Open Access Journals (Sweden)

    Ali Masri

    2017-01-01

    Full Text Available Multimodality requires the integration of heterogeneous transportation data to construct a broad view of the transportation network. Many new transportation services are emerging while being isolated from previously-existing networks. This leads them to publish their data sources to the web, according to linked data principles, in order to gain visibility. Our interest is to use these data to construct an extended transportation network that links these new services to existing ones. The main problems we tackle in this article fall in the categories of automatic schema matching and data interlinking. We propose an approach that uses web services as mediators to help in automatically detecting geospatial properties and mapping them between two different schemas. On the other hand, we propose a new interlinking approach that enables the user to define rich semantic links between datasets in a flexible and customizable way.

  15. Formation of a spatter-rich pyroclastic density current deposit in a Neogene sequence of trachytic-mafic igneous rocks at Mason Spur, Erebus volcanic province, Antarctica

    Science.gov (United States)

    Martin, A. P.; Smellie, J. L.; Cooper, A. F.; Townsend, D. B.

    2018-01-01

    Erosion has revealed a remarkable section through the heart of a volcanic island, Mason Spur, in the southwestern Ross Sea, Antarctica, including an unusually well-exposed section of caldera fill. The near-continuous exposure, 10 km laterally and > 1 km vertically, cuts through Cenozoic alkalic volcanic rocks of the Erebus volcanic province (McMurdo Volcanic Group) and permits the study of an ancient volcanic succession that is rarely available due to subsequent burial or erosion. The caldera filling sequence includes an unusual trachytic spatter-rich lapilli tuff (ignimbrite) facies that is particularly striking because of the presence of abundant black fluidal, dense juvenile spatter clasts of trachytic obsidian up to 2 m long supported in a pale cream-coloured pumiceous lapilli tuff matrix. Field mapping indicates that the deposit is an ignimbrite and, together with petrological considerations, it is suggested that mixing of dense spatter and pumiceous lapilli tuff in the investigated deposit occurred during emplacement, not necessarily in the same vent, with the mixed fragmental material emplaced as a pyroclastic density current. Liquid water was not initially present but a steam phase was probably generated during transport and may represent water ingested during passage of the current as it passed over either wet ground, stream, shallow lake or (possibly) snow. Well-exposed caldera interiors are uncommon and that at Mason Spur is helping understand eruption dynamics associated with a complex large island volcano. The results of our study should help to elucidate interpretations of other, less well exposed, pyroclastic density current deposits elsewhere in Antarctica and globally.

  16. C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

    Science.gov (United States)

    Kushwaha, Ambuj K; Grove, Anne

    2013-01-24

    Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

  17. Morphological character evolution of Amorphophallus (Araceae) based on a combined phylogenetic analysis of trnL, rbcL, and LEAFY second intron sequences

    NARCIS (Netherlands)

    Sedayu, A.; Eurlings, M.C.M.; Gravendeel, B.; Hetterscheid, W.L.A.

    2010-01-01

    Sequences of three different genes in 69 taxa of Amorphophallus were combined to reconstruct the molecular phylogeny of this species-rich Aroid genus. The data set was analyzed by three different methods, Maximum Parsimony, Maximum Likelihood and Bayesian analysis, producing slightly different tree

  18. Elementary martensitic transformation processes in Ni-rich NiTi single crystals with Ni4Ti3 precipitates

    International Nuclear Information System (INIS)

    Michutta, J.; Somsen, Ch.; Yawny, A.; Dlouhy, A.; Eggeler, G.

    2006-01-01

    The present study shows that multiple-step martensitic transformations can be observed in aged Ni-rich NiTi single crystals. Ageing of solution-annealed and water-quenched Ni-rich NiTi single crystals results in a homogeneous precipitation of coherent Ni 4 Ti 3 particles. When the interparticle spacing reaches a critical value (order of magnitude: 200 nm), three distinct transformation processes are observed on cooling from the high-temperature phase using differential scanning calorimetry and in situ transmission electron microscopy. The transformation sequence begins with the formation of R-phase starting from all precipitate/matrix interfaces (first step). The transformation continues with the formation of B19' and its subsequent growth along all precipitate/matrix interfaces (second step). Finally, the matrix in between the precipitates transforms to B19' (third step). Elementary transformation mechanisms which account for two- and three-step transformations in a system with small-scale microstructural heterogeneities were identified

  19. Looking Towards Curiosity's Canyon Path: a 4 km Sequence of Gully, Debris Deposits, and Fan/Deltas Which are Bordered by a Sloping Bedform-Capped Plain and Crossed by Lake Shorelines

    Science.gov (United States)

    Dietrich, W. E.; Palucis, M. C.; Parker, T.; Rubin, D.; dePablo, M. A.; Oehler, D. Z.; Bridges, N. T.

    2014-01-01

    The Curiosity Rover is headed towards layered outcrops that appear to be rich in phyllosilicates and sulphates with the expectation of an eventual ascent up Mt. Sharp. One likely will take the rover up a well-defined canyon. Inspection of CTX and HiRISE imagery and topography (5 m contour intervals) reveal a rich geomorphic sequence that may be encountered during the journey.

  20. A search for lithium-rich giant stars

    International Nuclear Information System (INIS)

    Brown, J.A.; Sneden, C.; Lambert, D.L.; Dutchover, E. Jr.

    1989-01-01

    Lithium abundances or upper limits have been determined for 644 bright G-K giant stars selected from the DDO photometric catalog. Two of these giants possess surface lithium abundances approaching the cosmic value of the interstellar medium and young main-sequence stars, and eight more giants have Li contents far in excess of standard predictions. At least some of these Li-rich giants are shown to be evolved to the stage of having convectively mixed envelopes, either from the direct evidence of low surface carbon isotope ratios, or from the indirect evidence of their H-R diagram positions. Suggestions are given for the unique conditions that might have allowed these stars to produce or accrete new lithium for their surface layers, or simply to preserve from destruction their initial lithium contents. The lithium abundance of the remaining stars demonstrates that giants only very rarely meet the expectations of standard first dredge-up theories; the average extra Li destruction required is about 1.5 dex. The evolutionary states of these giants and their average masses are discussed briefly, and the Li distribution of the giants is compared to predictions of Galactic chemical evolution. 110 refs