Spectroscopic and immunochemical characterization of left-handed Z RNA under physiological conditions

International Nuclear Information System (INIS)

Hardin, C.C.; Zarling, D.A.; Tinoco, I. Jr.

1986-01-01

Limited chemical brominination of poly[r(C-G)] results in partial modification of guanine C8 and cytosine C5 producing a mixture of A and Z RNA forms in 110 mM NaCl buffer at 37 0 C. 1 H NMR of Br-poly[r(C-G)] shows a 1:1 mixture of A and Z RNAs. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1' and CH1 resonances in both the A and Z forms. Distance estimates obtained from GH8 to GH1' NOEs demonstrate that even unbrominated guanine residues in these polynucleotides undergo a transition to the syn conformation (Z form). The Z form of Br-poly[r(C-G)] in 110 mM NaCl has a guanine Raman scattering band at 643 cm -1 in agreement with the band in the Z form of poly[r(C-G)] stabilized by 6 M NaClO 4 . In A RNA the band is at 671 cm -1 . The corresponding shift in DNA is from 682 cm -1 (B DNA) to 625 cm -1 (Z DNA). This suggests different glycosidic torsion angles in Z RNA and Z DNA. Inoculating rabbits with Br-poly[r(C-G)] produces polyclonal antibodies specific for the Z forms of RNA and DNA (i.e., poly[d(br 5 C-G)] or poly[d(io 5 C-G)] as judged by radioimmunoassay in 100 mM NaCl buffers. Phosphate buffer or certain high ionic strength conditions affect the recognition of Z RNA and Z DNA by anti-Z RNA or anti-Z DNA IgGs suggesting that one determinant recognized by these antibodies is the zig-zag phosphodiester backbone

RNA Catalysis, Thermodynamics and the Origin of Life

Directory of Open Access Journals (Sweden)

William G. Scott

2014-04-01

Full Text Available The RNA World Hypothesis posits that the first self-replicating molecules were RNAs. RNA self-replicases are, in general, assumed to have employed nucleotide 5ʹ-polyphosphates (or their analogues as substrates for RNA polymerization. The mechanism by which these substrates might be synthesized with sufficient abundance to supply a growing and evolving population of RNAs is problematic for evolutionary hypotheses because non-enzymatic synthesis and assembly of nucleotide 5ʹ-triphosphates (or other analogously activated phosphodiester species is inherently difficult. However, nucleotide 2ʹ,3ʹ-cyclic phosphates are also phosphodiesters, and are the natural and abundant products of RNA degradation. These have previously been dismissed as viable substrates for prebiotic RNA synthesis. We propose that the arguments for their dismissal are based on a flawed assumption, and that nucleotide 2ʹ,3ʹ-cyclic phosphates in fact possess several significant, advantageous properties that indeed make them particularly viable substrates for prebiotic RNA synthesis. An RNA World hypothesis based upon the polymerization of nucleotide 2ʹ,3ʹ-cyclic phosphates possesses additional explanatory power in that it accounts for the observed ribozyme “fossil record”, suggests a viable mechanism for substrate transport across lipid vesicle boundaries of primordial proto-cells, circumvents the problems of substrate scarcity and implausible synthetic pathways, provides for a primitive but effective RNA replicase editing mechanism, and definitively explains why RNA, rather than DNA, must have been the original catalyst. Finally, our analysis compels us to propose that a fundamental and universal property that drives the evolution of living systems, as well as pre-biotic replicating molecules (be they composed of RNA or protein, is that they exploit chemical reactions that already possess competing kinetically-preferred and thermodynamically-preferred pathways in a

On topological RNA interaction structures.

Science.gov (United States)

Qin, Jing; Reidys, Christian M

2013-07-01

Recently a folding algorithm of topological RNA pseudoknot structures was presented in Reidys et al. (2011). This algorithm folds single-stranded γ-structures, that is, RNA structures composed by distinct motifs of bounded topological genus. In this article, we set the theoretical foundations for the folding of the two backbone analogues of γ structures: the RNA γ-interaction structures. These are RNA-RNA interaction structures that are constructed by a finite number of building blocks over two backbones having genus at most γ. Combinatorial properties of γ-interaction structures are of practical interest since they have direct implications for the folding of topological interaction structures. We compute the generating function of γ-interaction structures and show that it is algebraic, which implies that the numbers of interaction structures can be computed recursively. We obtain simple asymptotic formulas for 0- and 1-interaction structures. The simplest class of interaction structures are the 0-interaction structures, which represent the two backbone analogues of secondary structures.

A New Experiment for the Measurement of nJ(C,P) Coupling Constants Including 3J(C4'i,Pi) and 3J(C4'i,Pi+1) in Oligonucleotides

International Nuclear Information System (INIS)

Richter, Christian; Reif, Bernd; Woerner, Karlheinz; Quant, Stefanie; Marino, John P.; Engels, Joachim W.; Griesinger, Christian; Schwalbe, Harald

1998-01-01

A new experiment for the measurement of nJ(C,P) coupling constants along the phosphodiester backbone in RNA and DNA based on a quantitative-J HCP experiment is presented. In addition to coupling constants, in which a carbon atom couples to only one phosphorus atom, both the intraresidual 3J(C4'i,Pi) and the sequential 3J(C4'i,Pi+1) for the C4' resonances that couple to two phosphorus atoms can be obtained. Coupling constants obtained by this new method are compared to values obtained from the P-FIDS experiment. Together with 3J(H,P) coupling constants measured using the P-FIDS experiment, the backbone angles β and element of can be determined

The DNA and RNA sugar-phosphate backbone emerges as the key player. An overview of quantum-chemical, structural biology and simulation studies

Czech Academy of Sciences Publication Activity Database

Šponer, Jiří; Mládek, Arnošt; Šponer, Judit E.; Svozil, Daniel; Zgarbová, M.; Banáš, Pavel; Jurečka, P.; Otyepka, M.

2012-01-01

Roč. 14, č. 44 (2012), s. 15257-15277 ISSN 1463-9076 R&D Projects: GA ČR(CZ) GD203/09/H046; GA ČR(CZ) GAP208/10/2302; GA ČR(CZ) GAP208/11/1822; GA ČR(CZ) GAP208/12/1878; GA ČR(CZ) GA203/09/1476; GA ČR(CZ) GBP305/12/G034 Institutional research plan: CEZ:AV0Z50040702 Keywords : DNA * RNA * sugar-phosphate backbone Subject RIV: BO - Biophysics Impact factor: 3.829, year: 2012

Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

Science.gov (United States)

Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

2013-01-01

The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

Science.gov (United States)

Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

2013-07-25

The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

Photolysis of phosphodiester bonds in plasmid DNA by high intensity UV laser irradiation

International Nuclear Information System (INIS)

Croke, D.T.; Blau, Werner; OhUigin, Colm; Kelly, J.M.; McConnell, D.J.

1988-01-01

The cleavage of phosphodiester bonds in DNA exposed to high intensity UV laser pulses in aerated aqueous solution has been investigated using a krypton fluoride excimer laser (248 nm) and bacterial plasmid DNA. The dependence of strand breakage on fluence and intensity has been studied in detail and shows that the process is non-linear with respect to intensity. The relationship between the quantum yield for strand breakage and intensity shows that the strand breakage reaction involves two-photon excitation of DNA bases. The quantum yield rises with intensity from a lower value of 7 x 10 -5 until a maximum value of 4.5 x 10 -4 is attained at intensities of 10 11 W m -2 and above. This value is approximately fifty-fold higher than the quantum yield for strand breakage induced by exposure to low density UV irradiation (254 nm, 12 W m -2 ). DNA sequencing experiments have shown that strand breakage occurs by the specific cleavage of the phosphodiester bond which lies immediately 3' to guanine residues in the DNA, leaving some alkali-labile remnant attached to the terminal phosphate. A mechanism for DNA strand breakage which involves the generation of guanine radical cations is proposed. (author)

Orientation-dependent backbone-only residue pair scoring functions for fixed backbone protein design

Directory of Open Access Journals (Sweden)

Bordner Andrew J

2010-04-01

Full Text Available Abstract Background Empirical scoring functions have proven useful in protein structure modeling. Most such scoring functions depend on protein side chain conformations. However, backbone-only scoring functions do not require computationally intensive structure optimization and so are well suited to protein design, which requires fast score evaluation. Furthermore, scoring functions that account for the distinctive relative position and orientation preferences of residue pairs are expected to be more accurate than those that depend only on the separation distance. Results Residue pair scoring functions for fixed backbone protein design were derived using only backbone geometry. Unlike previous studies that used spherical harmonics to fit 2D angular distributions, Gaussian Mixture Models were used to fit the full 3D (position only and 6D (position and orientation distributions of residue pairs. The performance of the 1D (residue separation only, 3D, and 6D scoring functions were compared by their ability to identify correct threading solutions for a non-redundant benchmark set of protein backbone structures. The threading accuracy was found to steadily increase with increasing dimension, with the 6D scoring function achieving the highest accuracy. Furthermore, the 3D and 6D scoring functions were shown to outperform side chain-dependent empirical potentials from three other studies. Next, two computational methods that take advantage of the speed and pairwise form of these new backbone-only scoring functions were investigated. The first is a procedure that exploits available sequence data by averaging scores over threading solutions for homologs. This was evaluated by applying it to the challenging problem of identifying interacting transmembrane alpha-helices and found to further improve prediction accuracy. The second is a protein design method for determining the optimal sequence for a backbone structure by applying Belief Propagation

Quantum Chemical Benchmark Study on 46 RNA Backbone Families Using a Dinucleotide Unit

Czech Academy of Sciences Publication Activity Database

Kruse, H.; Mládek, Arnošt; Gkionis, Konstantinos; Hansen, A.; Grimme, S.; Šponer, Jiří

2015-01-01

Roč. 11, č. 10 (2015), s. 4972-4991 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * DENSITY-FUNCTIONAL THEORY * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 5.301, year: 2015

Ribosomal proteins L11 and L10.(L12)4 and the antibiotic thiostrepton interact with overlapping regions of the 23 S rRNA backbone in the ribosomal GTPase centre

DEFF Research Database (Denmark)

Rosendahl, G; Douthwaite, S

1993-01-01

RNA, and to investigate how this interaction is influenced by other ribosomal components. Complexes were characterized in both naked 23 S rRNA and ribosomes from an E. coli L11-minus strain, before and after reconstitution with L11. The protein protects 17 riboses between positions 1058 and 1085 in the naked 23 S r......The Escherichia coli ribosomal protein (r-protein) L11 and its binding site on 23 S ribosomal RNA (rRNA) are associated with ribosomal hydrolysis of guanosine 5'-triphosphate (GTP). We have used hydroxyl radical footprinting to map the contacts between L11 and the backbone riboses in 23 S r......)4 and other proteins within the ribosome. The antibiotics thiostrepton and micrococcin inhibit the catalytic functions of this region by slotting in between the accessible loops and interacting with nucleotides there....

Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus

International Nuclear Information System (INIS)

Gassner, G.T.; Dickie, J.P.; Hamerski, D.A.; Magnuson, J.K.; Anderson, J.S.

1990-01-01

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31 P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB 3 H 4 followed by complete acid hydrolysis yielded [ 3 H] glucosaminitol from the true reducing end residue of teichuronic acid and [ 3 H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue

Testing Backbone.js

CERN Document Server

Roemer, Ryan

2013-01-01

This book is packed with the step by step tutorial and instructions in recipe format helping you setup test infrastructure and gradually advance your skills to plan, develop, and test your backbone applications.If you are a JavaScript developer looking for recipes to create and implement test support for your backbone application, then this book is ideal for you.

1990s: High Capacity Backbones

Indian Academy of Sciences (India)

First page Back Continue Last page Overview Graphics. 1990s: High Capacity Backbones. Backbone capacities increased from 2.5 Gb/s to 100s of Gb/s during the 1990's. Wavelength division multiplexing with 160 waves of 10 Gb/s was commercially available. Several high-capacity backbones built in the US and Europe.

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...

Controlling the structure of sequence-defined poly(phosphodiester)s for optimal MS/MS reading of digital information.

Science.gov (United States)

Amalian, J-A; Al Ouahabi, A; Cavallo, G; König, N F; Poyer, S; Lutz, J-F; Charles, L

2017-11-01

Digital polymers are monodisperse chains with a controlled sequence of co-monomers, defined as letters of an alphabet, and are used to store information at the molecular level. Reading such messages is hence a sequencing task that can be efficiently achieved by tandem mass spectrometry. To improve their readability, structure of sequence-controlled synthetic polymers can be optimized, based on considerations regarding their fragmentation behavior. This strategy is described here for poly(phosphodiester)s, which were synthesized as monodisperse chains with more than 100 units but exhibited extremely complex dissociation spectra. In these polymers, two repeating units that differ by a simple H/CH 3 variation were defined as the 0 and 1 bit of the ASCII code and spaced by a phosphate moiety. They were readily ionized in negative ion mode electrospray but dissociated via cleavage at all phosphate bonds upon collisional activation. Although allowing a complete sequence coverage of digital poly(phosphodiester)s, this fragmentation behavior was not efficient for macromolecules with more than 50 co-monomers, and data interpretation was very tedious. The structure of these polymers was then modified by introducing alkoxyamine linkages at appropriate location throughout the chain. A first design consisted of placing these low dissociation energy bonds between each monomeric bit: while cleavage of this sole bond greatly simplified MS/MS spectra, efficient sequencing was limited to chains with up to about 50 units. In contrast, introduction of alkoxyamine bonds between each byte (i.e. a set of eight co-monomers) was a more successful strategy. Long messages (so far, up to 8 bytes) could be read in MS 3 experiments, where single-byte containing fragments released during the first activation stage were further dissociated for sequencing. The whole sequence of such byte-truncated poly(phosphodiester)s could be easily re-constructed based on a mass tagging system which permits

Characteristics of enzyme hydrolyzing natural covalent bond between RNA and protein VPg of encephalomyocarditis virus

International Nuclear Information System (INIS)

Drygin, Yu.F.; Siyanova, E.Yu.

1986-01-01

The isolation and a preliminary characterization of the enzyme specifically hydrolyzing the phosphodiester bond between protein VPg and the RNA of encephalomyocarditis virus was the goal of the present investigation. The enzyme was isolated from a salt extract of Krebs II mouse ascites carcinoma cells by ion-exchange and affinity chromatography. It was found that the enzyme actually specifically cleaves the covalent bond between the RNA and protein, however, the isolation procedure does not free the enzyme from impurities which partially inhibit it. The enzyme cleaves the RNA-protein VPg complex of polio virus at a high rate, it is completely inactivated at 55 0 C, and is partially inhibited by EDTA

Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers

DEFF Research Database (Denmark)

Hansen, A C; Grunwald, T; Lund, Anders Henrik

2001-01-01

could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold...

RCrane: semi-automated RNA model building.

Science.gov (United States)

Keating, Kevin S; Pyle, Anna Marie

2012-08-01

RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems.

Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

OpenAIRE

Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

2013-01-01

The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This mo...

Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3'-5')thymidine.

Science.gov (United States)

Swenson, D H; Lawley, P D

1978-01-01

1. The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates. PMID:208508

Backbone upgrades and DEC equipment replacement

Science.gov (United States)

Vancamp, Warren

1991-01-01

The NASA Science Internet (NSI) dual protocol backbone is outlined. It includes DECnet link upgrades to match TCP/IP link performance. It also includes the integration of backbone resources and central management. The phase 1 transition process is outlined.

High Performance Infiltrated Backbones for Cathode-Supported SOFC's

DEFF Research Database (Denmark)

Gil, Vanesa; Kammer Hansen, Kent

2014-01-01

The concept of using highly ionic conducting backbones with subsequent infiltration of electronically conducting particles has widely been used to develop alternative anode-supported SOFC's. In this work, the idea was to develop infiltrated backbones as an alternative design based on cathode......, microstructural characterization and electrochemical testing are discussed. Data on polarization resistance, Rp, are obtained from impedance spectra recorded on quasi-symmetrical cells (YSZ backbones/YSZ/LSM-YSZ (screen printed)). The backbones are infiltrated with LSM and compared to a standard LSM-YSZ screen...

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs.

Science.gov (United States)

Mutisya, Daniel; Hardcastle, Travis; Cheruiyot, Samwel K; Pallan, Pradeep S; Kennedy, Scott D; Egli, Martin; Kelley, Melissa L; Smith, Anja van Brabant; Rozners, Eriks

2017-08-21

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA-DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Porous solid backbone impregnation for electrochemical energy conversion systems

KAUST Repository

Boulfrad, Samir; Jabbour, Ghassan

2013-01-01

An apparatus and method for impregnating a porous solid backbone. The apparatus may include a platform for holding a porous solid backbone, an ink jet nozzle configured to dispense a liquid solution onto the porous solid backbone, a positioning mechanism configured to position the ink jet nozzle proximate to a plurality of locations of the porous solid backbone, and a control unit configured to control the positioning mechanism to position the ink jet nozzle proximate to the plurality of locations and cause the ink jet nozzle to dispense the liquid solution onto the porous solid backbone.

Porous solid backbone impregnation for electrochemical energy conversion systems

KAUST Repository

Boulfrad, Samir

2013-09-19

An apparatus and method for impregnating a porous solid backbone. The apparatus may include a platform for holding a porous solid backbone, an ink jet nozzle configured to dispense a liquid solution onto the porous solid backbone, a positioning mechanism configured to position the ink jet nozzle proximate to a plurality of locations of the porous solid backbone, and a control unit configured to control the positioning mechanism to position the ink jet nozzle proximate to the plurality of locations and cause the ink jet nozzle to dispense the liquid solution onto the porous solid backbone.

A new RNA branching activity: the GIR1 ribozyme

DEFF Research Database (Denmark)

Nielsen, Henrik; Johansen, Steinar D

2006-01-01

The formation of lariat intermediates during the first step of splicing of group II introns and spliceosomal introns is a well-studied fundamental reaction in molecular biology. Apart from this prominent example, there are surprisingly few occurrences of branched nucleotides or even 2......',5'-phosphodiester bonds in biology. We recently described a new ribozyme, the GIR1 branching ribozyme, which catalyzes the formation of a tiny lariat that caps an mRNA. This new example together with work on artificial branching ribozymes and deoxyribozymes shows that branching is facile and points...... to the possibility that branching reactions could be more prevalent than previously recognized....

DBR1 siRNA inhibition of HIV-1 replication

Directory of Open Access Journals (Sweden)

Naidu Yathi

2005-10-01

Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

High-resolution protein design with backbone freedom.

Science.gov (United States)

Harbury, P B; Plecs, J J; Tidor, B; Alber, T; Kim, P S

1998-11-20

Recent advances in computational techniques have allowed the design of precise side-chain packing in proteins with predetermined, naturally occurring backbone structures. Because these methods do not model protein main-chain flexibility, they lack the breadth to explore novel backbone conformations. Here the de novo design of a family of alpha-helical bundle proteins with a right-handed superhelical twist is described. In the design, the overall protein fold was specified by hydrophobic-polar residue patterning, whereas the bundle oligomerization state, detailed main-chain conformation, and interior side-chain rotamers were engineered by computational enumerations of packing in alternate backbone structures. Main-chain flexibility was incorporated through an algebraic parameterization of the backbone. The designed peptides form alpha-helical dimers, trimers, and tetramers in accord with the design goals. The crystal structure of the tetramer matches the designed structure in atomic detail.

Optical burst switching based satellite backbone network

Science.gov (United States)

Li, Tingting; Guo, Hongxiang; Wang, Cen; Wu, Jian

2018-02-01

We propose a novel time slot based optical burst switching (OBS) architecture for GEO/LEO based satellite backbone network. This architecture can provide high speed data transmission rate and high switching capacity . Furthermore, we design the control plane of this optical satellite backbone network. The software defined network (SDN) and network slice (NS) technologies are introduced. Under the properly designed control mechanism, this backbone network is flexible to support various services with diverse transmission requirements. Additionally, the LEO access and handoff management in this network is also discussed.

RCrane: semi-automated RNA model building

International Nuclear Information System (INIS)

Keating, Kevin S.; Pyle, Anna Marie

2012-01-01

RCrane is a new tool for the partially automated building of RNA crystallographic models into electron-density maps of low or intermediate resolution. This tool helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs

Energy Technology Data Exchange (ETDEWEB)

Mutisya, Daniel; Hardcastle, Travis; Cheruiyot, Samwel K.; Pallan, Pradeep S.; Kennedy, Scott D.; Egli, Martin; Kelley, Melissa L.; Smith, Anja van Brabant; Rozners, Eriks

2017-06-27

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA–DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs.

NMR studies of abasic sites in DNA duplexes: Deoxyadenosine stacks into the helix opposite acyclic lesions

International Nuclear Information System (INIS)

Kalnik, M.W.; Chang, Chienneng; Johnson, F.; Grollman, A.P.; Patel, D.J.

1989-01-01

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C)·d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated AP P 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C-)·d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated AP E 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the AP P 9-mer and AP E 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H 2 O and D 2 O solution between -5 and 5 degree C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and El14) and stacks between the flanking G4·C15 and G6·C13 Watson-Crick base pairs in both the AP P 9-mer and AP E 9-mer duplexes. Proton NMR parameters for the Ap P 9-mer and AP E 9-mer duplexes are similar to those reported previously. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the AP P 9-mer and AP E 9-mer duplexes (5 degree C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles

High Performance Infiltrated Backbones for Cathode-Supported SOFC's

DEFF Research Database (Denmark)

Gil, Vanesa; Kammer Hansen, Kent

2014-01-01

A four-step infiltration method has been developed to infiltrate La0.75Sr0.25MnO3+δ (LSM25) nanoparticles into porous structures (YSZ or LSM-YSZ backbones). The pore size distribution in the backbones is obtained either by using PMMA and/or graphites as pore formers or by leaching treatment of sa...... of samples with Ni remained in the YSZ structure at high temperatures. All impregnated backbones, presented Rs comparable to a standard screen printed cathode, which proves that LSM nanoparticles forms a pathway for electron conduction....

Radiation safety system (RSS) backbones: Design, engineering, fabrication and installation

International Nuclear Information System (INIS)

Wilmarth, J.E.; Sturrock, J.C.; Gallegos, F.R.

1998-01-01

The Radiation Safety System (RSS) Backbones are part of an electrical/electronic/mechanical system insuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS Backbones control the safety fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low energy beam transport. The Backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the Backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two Linac Backbone segments and experimental area segments form a continuous cable plant over 3,500 feet from beam plugs to the tip on the longest tail. The Backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely

Cost-effectiveness analysis of dolutegravir plus backbone compared with raltegravir plus backbone, darunavir+ritonavir plus backbone and efavirenz/tenofovir/emtricitabine in treatment naïve and experienced HIV-positive patients

Directory of Open Access Journals (Sweden)

Restelli U

2017-06-01

Full Text Available Umberto Restelli,1,2 Giuliano Rizzardini,3,4 Andrea Antinori,5 Adriano Lazzarin,6 Marzia Bonfanti,1 Paolo Bonfanti,7 Davide Croce1,2 1Centre for Research on Health Economics, Social and Health Care Management, LIUC – Università Cattaneo, Castellanza, Varese, Italy; 2School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 3First and Second Divisions of Infectious Diseases, “Luigi Sacco” Hospital, Milan, Italy; 4School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 5National Institute for Infectious Diseases “L Spallanzani”, Rome, 6Department of Infectious Diseases, San Raffaele Scientific Institute, 7Department of Infectious and Tropical Diseases, A Manzoni Hospital, Lecco, Italy Background: In January 2014, the European Medicines Agency issued a marketing authorization for dolutegravir (DTG, a second-generation integrase strand transfer inhibitor for HIV treatment. The study aimed at determining the incremental cost-effectiveness ratio (ICER of the use of DTG+backbone compared with raltegravir (RAL+backbone, darunavir (DRV+ritonavir(r+backbone and efavirenz/tenofovir/emtricitabine (EFV/TDF/FTC in HIV-positive treatment-naïve patients and compared with RAL+backbone in treatment-experienced patients, from the Italian National Health Service’s point of view.Materials and methods: A published Monte Carlo Individual Simulation Model (ARAMIS-DTG model was used to perform the analysis. Patients pass through mutually exclusive health states (defined in terms of diagnosis of HIV with or without opportunistic infections [OIs] and cardiovascular disease [CVD] and successive lines of therapy. The model considers costs (2014 and quality of life per monthly cycle in a lifetime horizon. Costs and quality-adjusted life years (QALYs are dependent on OI, CVD, AIDS events, adverse events and antiretroviral therapies.Results: In

High-Throughput Sequencing Based Methods of RNA Structure Investigation

DEFF Research Database (Denmark)

Kielpinski, Lukasz Jan

In this thesis we describe the development of four related methods for RNA structure probing that utilize massive parallel sequencing. Using them, we were able to gather structural data for multiple, long molecules simultaneously. First, we have established an easy to follow experimental...... and computational protocol for detecting the reverse transcription termination sites (RTTS-Seq). This protocol was subsequently applied to hydroxyl radical footprinting of three dimensional RNA structures to give a probing signal that correlates well with the RNA backbone solvent accessibility. Moreover, we applied...

Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

Science.gov (United States)

Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

2016-01-01

Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

Evidence from in vivo 31-phosphorus magnetic resonance spectroscopy phosphodiesters that exhaled ethane is a biomarker of cerebral n-3 polyunsaturated fatty acid peroxidation in humans

Directory of Open Access Journals (Sweden)

Hamilton Gavin

2008-04-01

Full Text Available Abstract Background This study tested the hypothesis that exhaled ethane is a biomarker of cerebral n-3 polyunsaturated fatty acid peroxidation in humans. Ethane is released specifically following peroxidation of n-3 polyunsaturated fatty acids. We reasoned that the cerebral source of ethane would be the docosahexaenoic acid component of membrane phospholipids. Breakdown of the latter also releases phosphorylated polar head groups, giving rise to glycerophosphorylcholine and glycerophosphorylethanolamine, which can be measured from the 31-phosphorus neurospectroscopy phosphodiester peak. Schizophrenia patients were chosen because of evidence of increased free radical-mediated damage and cerebral lipid peroxidation in this disorder. Methods Samples of alveolar air were obtained from eight patients and ethane was analyzed and quantified by gas chromatography and mass spectrometry (m/z = 30. Cerebral 31-phosphorus spectra were obtained from the same patients at a magnetic field strength of 1.5 T using an image-selected in vivo spectroscopy sequence (TR = 10 s; 64 signal averages localized on a 70 × 70 × 70 mm3 voxel. The quantification of the 31-phosphorus signals using prior knowledge was carried out in the temporal domain after truncating the first 1.92 ms of the signal to remove the broad component present in the 31-phosphorus spectra. Results The ethane and phosphodiester levels, expressed as a percentage of the total 31-phosphorus signal, were positively and significantly correlated (rs = 0.714, p Conclusion Our results support the hypothesis that the measurement of exhaled ethane levels indexes cerebral n-3 lipid peroxidation. From a practical viewpoint, if human cerebral n-3 polyunsaturated fatty acid catabolism can be measured by ethane in expired breath, this would be more convenient than determining the area of the 31-phosphorus neurospectroscopy phosphodiester peak.

Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

Science.gov (United States)

Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

2016-03-17

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. Copyright © 2016 Elsevier Inc. All rights reserved.

APPECT: An Approximate Backbone-Based Clustering Algorithm for Tags

DEFF Research Database (Denmark)

Zong, Yu; Xu, Guandong; Jin, Pin

2011-01-01

algorithm for Tags (APPECT). The main steps of APPECT are: (1) we execute the K-means algorithm on a tag similarity matrix for M times and collect a set of tag clustering results Z={C1,C2,…,Cm}; (2) we form the approximate backbone of Z by executing a greedy search; (3) we fix the approximate backbone...... as the initial tag clustering result and then assign the rest tags into the corresponding clusters based on the similarity. Experimental results on three real world datasets namely MedWorm, MovieLens and Dmoz demonstrate the effectiveness and the superiority of the proposed method against the traditional...... Agglomerative Clustering on tagging data, which possess the inherent drawbacks, such as the sensitivity of initialization. In this paper, we instead make use of the approximate backbone of tag clustering results to find out better tag clusters. In particular, we propose an APProximate backbonE-based Clustering...

A rapid NMR-based method for discrimination of strain-specific cell wall teichoic acid structures reveals a third backbone type in Lactobacillus plantarum.

Science.gov (United States)

Tomita, Satoru; Tanaka, Naoto; Okada, Sanae

2017-03-01

The lactic acid bacterium Lactobacillus plantarum is capable of producing strain-specific structures of cell wall teichoic acid (WTA), an anionic polysaccharide found in the Gram-positive bacterial cell wall. In this study, we established a rapid, NMR-based procedure to discriminate WTA structures in this species, and applied it to 94 strains of L. plantarum. Six previously reported glycerol- and ribitol-containing WTA subtypes were successfully identified from 78 strains, suggesting that these were the dominant structures. However, the level of structural variety differed markedly among bacterial sources, possibly reflecting differences in strain-level microbial diversity. WTAs from eight strains were not identified based on NMR spectra and were classified into three groups. Structural analysis of a partial degradation product of an unidentified WTA produced by strain TUA 1496L revealed that the WTA was 1-O-β-d-glucosylglycerol. Two-dimensional NMR analysis of the polymer structure showed phosphodiester bonds between C-3 and C-6 of the glycerol and glucose residues, suggesting a polymer structure of 3,6΄-linked poly(1-O-β-d-glucosyl-sn-glycerol phosphate). This is the third WTA backbone structure in L. plantarum, following 3,6΄-linked poly(1-O-α-d-glucosyl-sn-glycerol phosphate) and 1,5-linked poly(ribitol phosphate). © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Adding diverse noncanonical backbones to rosetta: enabling peptidomimetic design.

Directory of Open Access Journals (Sweden)

Kevin Drew

Full Text Available Peptidomimetics are classes of molecules that mimic structural and functional attributes of polypeptides. Peptidomimetic oligomers can frequently be synthesized using efficient solid phase synthesis procedures similar to peptide synthesis. Conformationally ordered peptidomimetic oligomers are finding broad applications for molecular recognition and for inhibiting protein-protein interactions. One critical limitation is the limited set of design tools for identifying oligomer sequences that can adopt desired conformations. Here, we present expansions to the ROSETTA platform that enable structure prediction and design of five non-peptidic oligomer scaffolds (noncanonical backbones, oligooxopiperazines, oligo-peptoids, [Formula: see text]-peptides, hydrogen bond surrogate helices and oligosaccharides. This work is complementary to prior additions to model noncanonical protein side chains in ROSETTA. The main purpose of our manuscript is to give a detailed description to current and future developers of how each of these noncanonical backbones was implemented. Furthermore, we provide a general outline for implementation of new backbone types not discussed here. To illustrate the utility of this approach, we describe the first tests of the ROSETTA molecular mechanics energy function in the context of oligooxopiperazines, using quantum mechanical calculations as comparison points, scanning through backbone and side chain torsion angles for a model peptidomimetic. Finally, as an example of a novel design application, we describe the automated design of an oligooxopiperazine that inhibits the p53-MDM2 protein-protein interaction. For the general biological and bioengineering community, several noncanonical backbones have been incorporated into web applications that allow users to freely and rapidly test the presented protocols (http://rosie.rosettacommons.org. This work helps address the peptidomimetic community's need for an automated and expandable

Green Network Planning Model for Optical Backbones

DEFF Research Database (Denmark)

Gutierrez Lopez, Jose Manuel; Riaz, M. Tahir; Jensen, Michael

2010-01-01

on the environment in general. In network planning there are existing planning models focused on QoS provisioning, investment minimization or combinations of both and other parameters. But there is a lack of a model for designing green optical backbones. This paper presents novel ideas to be able to define......Communication networks are becoming more essential for our daily lives and critically important for industry and governments. The intense growth in the backbone traffic implies an increment of the power demands of the transmission systems. This power usage might have a significant negative effect...

Determination of backbone chain direction of PDA using FFM

Science.gov (United States)

Jo, Sadaharu; Okamoto, Kentaro; Takenaga, Mitsuru

2010-01-01

The effect of backbone chains on friction force was investigated on both Langmuir-Blodgett (LB) films of 10,12-heptacosadiynoic acid and the (0 1 0) surfaces of single crystals of 2,4-hexadiene-1,6-diol using friction force microscopy (FFM). It was observed that friction force decreased when the scanning direction was parallel to the [0 0 1] direction in both samples. Moreover, friction force decreased when the scanning direction was parallel to the crystallographic [1 0 2], [1 0 1], [1 0 0] and [1 0 1¯] directions in only the single crystals. For the LB films, the [0 0 1] direction corresponds to the backbone chain direction of 10,12-heptacosadiynoic acid. For the single crystals, both the [0 0 1] and [1 0 1] directions correspond to the backbone chain direction, and the [1 0 2], [1 0 0] and [1 0 1¯] directions correspond to the low-index crystallographic direction. In both the LB films and single crystals, the friction force was minimized when the directions of scanning and the backbone chain were parallel.

Screening of Modified RNA duplexes

DEFF Research Database (Denmark)

Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form......Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...

On Backbone Structure for a Future Multipurpose Network

DEFF Research Database (Denmark)

Gutierrez Lopez, Jose Manuel; Cuevas, Ruben; Riaz, M. Tahir

2008-01-01

Telecommunications are evolving towards the unification of services and infrastructures. This unification must be achieved at the highest hierarchical level for a complete synergy of services. Therefore, one of the requirements is a multipurpose backbone network capable of supporting all the curr......Telecommunications are evolving towards the unification of services and infrastructures. This unification must be achieved at the highest hierarchical level for a complete synergy of services. Therefore, one of the requirements is a multipurpose backbone network capable of supporting all...

Solution, solid phase and computational structures of apicidin and its backbone-reduced analogs.

Science.gov (United States)

Kranz, Michael; Murray, Peter John; Taylor, Stephen; Upton, Richard J; Clegg, William; Elsegood, Mark R J

2006-06-01

The recently isolated broad-spectrum antiparasitic apicidin (1) is one of the few naturally occurring cyclic tetrapeptides (CTP). Depending on the solvent, the backbone of 1 exhibits two gamma-turns (in CH(2)Cl(2)) or a beta-turn (in DMSO), differing solely in the rotation of the plane of one of the amide bonds. In the X-ray crystal structure, the peptidic C==Os and NHs are on opposite sides of the backbone plane, giving rise to infinite stacks of cyclotetrapeptides connected by three intermolecular hydrogen bonds between the backbones. Conformational searches (Amber force field) on a truncated model system of 1 confirm all three backbone conformations to be low-energy states. The previously synthesized analogs of 1 containing a reduced amide bond exhibit the same backbone conformation as 1 in DMSO, which is confirmed further by the X-ray crystal structure of a model system of the desoxy analogs of 1. This similarity helps in explaining why the desoxy analogs retain some of the antiprotozoal activities of apicidin. The backbone-reduction approach designed to facilitate the cyclization step of the acyclic precursors of the CTPs seems to retain the conformational preferences of the parent peptide backbone.

Molding a peptide into an RNA site by in vivo peptide evolution

OpenAIRE

Harada, Kazuo; Martin, Shelley S.; Tan, Ruoying; Frankel, Alan D.

1997-01-01

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been ...

Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

Directory of Open Access Journals (Sweden)

Colin A Smith

Full Text Available Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface, interactions between and within parts of the structure (e.g. domains can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

ExScal Backbone Network Architecture

Science.gov (United States)

2005-01-01

802.11 battery powered nodes was laid over the sensor network. We adopted the Stargate platform for the backbone tier to serve as the basis for...its head. XSS Hardware and Network: XSS stands for eXtreme Scaling Stargate . A stargate is a linux-based single board computer. It has a 400 MHz

Underestimated Halogen Bonds Forming with Protein Backbone in Protein Data Bank.

Science.gov (United States)

Zhang, Qian; Xu, Zhijian; Shi, Jiye; Zhu, Weiliang

2017-07-24

Halogen bonds (XBs) are attracting increasing attention in biological systems. Protein Data Bank (PDB) archives experimentally determined XBs in biological macromolecules. However, no software for structure refinement in X-ray crystallography takes into account XBs, which might result in the weakening or even vanishing of experimentally determined XBs in PDB. In our previous study, we showed that side-chain XBs forming with protein side chains are underestimated in PDB on the basis of the phenomenon that the proportion of side-chain XBs to overall XBs decreases as structural resolution becomes lower and lower. However, whether the dominant backbone XBs forming with protein backbone are overlooked is still a mystery. Here, with the help of the ratio (R F ) of the observed XBs' frequency of occurrence to their frequency expected at random, we demonstrated that backbone XBs are largely overlooked in PDB, too. Furthermore, three cases were discovered possessing backbone XBs in high resolution structures while losing the XBs in low resolution structures. In the last two cases, even at 1.80 Å resolution, the backbone XBs were lost, manifesting the urgent need to consider XBs in the refinement process during X-ray crystallography study.

The Graphical Representation of the Digital Astronaut Physiology Backbone

Science.gov (United States)

Briers, Demarcus

2010-01-01

This report summarizes my internship project with the NASA Digital Astronaut Project to analyze the Digital Astronaut (DA) physiology backbone model. The Digital Astronaut Project (DAP) applies integrated physiology models to support space biomedical operations, and to assist NASA researchers in closing knowledge gaps related to human physiologic responses to space flight. The DA physiology backbone is a set of integrated physiological equations and functions that model the interacting systems of the human body. The current release of the model is HumMod (Human Model) version 1.5 and was developed over forty years at the University of Mississippi Medical Center (UMMC). The physiology equations and functions are scripted in an XML schema specifically designed for physiology modeling by Dr. Thomas G. Coleman at UMMC. Currently it is difficult to examine the physiology backbone without being knowledgeable of the XML schema. While investigating and documenting the tags and algorithms used in the XML schema, I proposed a standard methodology for a graphical representation. This standard methodology may be used to transcribe graphical representations from the DA physiology backbone. In turn, the graphical representations can allow examination of the physiological functions and equations without the need to be familiar with the computer programming languages or markup languages used by DA modeling software.

Interaction of sulforaphane with DNA and RNA.

Directory of Open Access Journals (Sweden)

Farzaneh Abassi Joozdani

Full Text Available Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN-DNA and -RNA complexes by Fourier transform infrared (FTIR and UV-Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2, while RNA binding is through G, U, A bases with some degree of SFN-phosphate (PO2 interaction. Overall binding constants were estimated to be K(SFN-DNA=3.01 (± 0.035×10(4 M(-1 and K(SFN-RNA= 6.63 (±0.042×10(3 M(-1. At high SFN concentration (SFN/RNA = 1/1, DNA conformation changed from B to A occurred, while RNA remained in A-family structure.

Backbone Diversity Analysis in Catalyst Design

NARCIS (Netherlands)

Maldonado, A.G.; Hageman, J.A.; Mastroianni, S.; Rothenberg, G.

2009-01-01

We present a computer-based heuristic framework for designing libraries of homogeneous catalysts. In this approach, a set of given bidentate ligand-metal complexes is disassembled into key substructures (building blocks). These include metal atoms, ligating groups, backbone groups, and residue

Exact Solutions for Internuclear Vectors and Backbone Dihedral Angles from NH Residual Dipolar Couplings in Two Media, and their Application in a Systematic Search Algorithm for Determining Protein Backbone Structure

International Nuclear Information System (INIS)

Wang Lincong; Donald, Bruce Randall

2004-01-01

We have derived a quartic equation for computing the direction of an internuclear vector from residual dipolar couplings (RDCs) measured in two aligning media, and two simple trigonometric equations for computing the backbone (φ,ψ) angles from two backbone vectors in consecutive peptide planes. These equations make it possible to compute, exactly and in constant time, the backbone (φ,ψ) angles for a residue from RDCs in two media on any single backbone vector type. Building upon these exact solutions we have designed a novel algorithm for determining a protein backbone substructure consisting of α-helices and β-sheets. Our algorithm employs a systematic search technique to refine the conformation of both α-helices and β-sheets and to determine their orientations using exclusively the angular restraints from RDCs. The algorithm computes the backbone substructure employing very sparse distance restraints between pairs of α-helices and β-sheets refined by the systematic search. The algorithm has been demonstrated on the protein human ubiquitin using only backbone NH RDCs, plus twelve hydrogen bonds and four NOE distance restraints. Further, our results show that both the global orientations and the conformations of α-helices and β-strands can be determined with high accuracy using only two RDCs per residue. The algorithm requires, as its input, backbone resonance assignments, the identification of α-helices and β-sheets as well as sparse NOE distance and hydrogen bond restraints.Abbreviations: NMR - nuclear magnetic resonance; RDC - residual dipolar coupling; NOE - nuclear Overhauser effect; SVD - singular value decomposition; DFS - depth-first search; RMSD - root mean square deviation; POF - principal order frame; PDB - protein data bank; SA - simulated annealing; MD - molecular dynamics

Thin Films Formed from Conjugated Polymers with Ionic, Water-Soluble Backbones

NARCIS (Netherlands)

Voortman, Thomas P; Chiechi, Ryan C

2015-01-01

This paper compares the morphologies of films of conjugated polymers in which the backbone (main chain) and pendant groups are varied between ionic/hydrophilic and aliphatic/hydrophobic. We observe that conjugated polymers in which the pendant groups and backbone are matched, either ionic-ionic or

Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs

Science.gov (United States)

Mutisya, Daniel; Selvam, Chelliah; Lunstad, Benjamin D.; Pallan, Pradeep S.; Haas, Amanda; Leake, Devin; Egli, Martin; Rozners, Eriks

2014-01-01

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3′-CH2-CO-NH-5′ amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P–OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5′-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications. PMID:24813446

Nonribosomal biosynthesis of backbone-modified peptides

Science.gov (United States)

Niquille, David L.; Hansen, Douglas A.; Mori, Takahiro; Fercher, David; Kries, Hajo; Hilvert, Donald

2018-03-01

Biosynthetic modification of nonribosomal peptide backbones represents a potentially powerful strategy to modulate the structure and properties of an important class of therapeutics. Using a high-throughput assay for catalytic activity, we show here that an L-Phe-specific module of an archetypal nonribosomal peptide synthetase can be reprogrammed to accept and process the backbone-modified amino acid (S)-β-Phe with near-native specificity and efficiency. A co-crystal structure with a non-hydrolysable aminoacyl-AMP analogue reveals the origins of the 40,000-fold α/β-specificity switch, illuminating subtle but precise remodelling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-β-Phe-containing peptides were produced at preparative scale in vitro (~1 mmol) and high titres in vivo (~100 mg l-1), highlighting the potential of biosynthetic pathway engineering for the construction of novel nonribosomal β-frameworks.

Gender features of functional condition of backbone of teenagers with scoliotic posture

Directory of Open Access Journals (Sweden)

Sergiy Afanasiev

2016-10-01

Full Text Available Purpose: to study mobility of backbone, endurance of muscles of a trunk and to define gender features of functional condition of backbone at children of the middle school age with scoliotic posture depending on the direction of the top of arch of curvature of spine. Material & Methods: 40 girls and 40 boys, including 18 girls and 18 boys with the right-side deformation of backbone in the thoracic department, the left-side – 22 girls and 22 boys are examined. Results: features of changes of indicators, depending on sex of children and frontage of the top of arch of curvature of spine column, are revealed when studying the level of flexibility of backbone and endurance of muscles of a trunk at children of the middle school age with scoliotic posture. Conclusions: it is established that the level of decrease in flexibility of backbone is higher at boys, than at girls, whereas indicators of contractile ability and tone of muscles of "muscular corset" are higher at boys.

Customization of Artificial MicroRNA Design.

Science.gov (United States)

Van Vu, Tien; Do, Vinh Nang

2017-01-01

RNAi approaches, including microRNA (miRNA) regulatory pathway, offer great tools for functional characterization of unknown genes. Moreover, the applications of artificial microRNA (amiRNA) in the field of plant transgenesis have also been advanced to engineer pathogen-resistant or trait-improved transgenic plants. Until now, despite the high potency of amiRNA approach, no commercial plant cultivar expressing amiRNAs with improved traits has been released yet. Beside the issues of biosafety policies, the specificity and efficacy of amiRNAs are of major concerns. Sufficient cares should be taken for the specificity and efficacy of amiRNAs due to their potential off-target effects and other issues relating to in vivo expression of pre-amiRNAs. For these reasons, the proper design of amiRNAs with the lowest off-target possibility is very important for successful applications of the approach in plant. Therefore, there are many studies with the aim to improve the amiRNA design and amiRNA expressing backbones for obtaining better specificity and efficacy. However, the requirement for an efficient reference for the design is still needed. In the present chapter, we attempt to summarize and discuss all the major concerns relating to amiRNA design with the hope to provide a significant guideline for this approach.

Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone.

Science.gov (United States)

Gui, Meng; Song, Juyi; Zhang, Lu; Wang, Shun; Wu, Ruiyun; Ma, Changwei; Li, Pinglan

2015-06-05

Chondroitin sulfates (CSs) were extracted from sturgeon skull and backbone, and their chemical composition, anticoagulant, anti-platelet and thrombolysis activities were evaluated. The average molecular weights of CS from sturgeon skull and backbone were 38.5kDa and 49.2kDa, respectively. Disaccharide analysis indicated that the sturgeon backbone CS was primarily composed of disaccharide monosulfated in position four of the GalNAc (37.8%) and disaccharide monosulfated in position six of the GalNAc (59.6%) while sturgeon skull CS was primarily composed of nonsulfated disaccharide (74.2%). Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS. Sturgeon backbone CS could significantly prolong activated partial thromboplastin time (APTT) and thrombin time (TT), inhibited ADP-induced platelet aggregation and dissolved platelet plasma clots in vitro. The results suggested that sturgeon backbone CS can be explored as a functional food with antithrombotic function. Copyright © 2015 Elsevier Ltd. All rights reserved.

tRNA conjugation with chitosan nanoparticles: An AFM imaging study.

Science.gov (United States)

Agudelo, D; Kreplak, L; Tajmir-Riahi, H A

2016-04-01

The conjugation of tRNA with chitosan nanoparticles of different sizes 15,100 and 200 kDa was investigated in aqueous solution using multiple spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed that chitosan binds tRNA via G-C and A-U base pairs as well as backbone PO2 group, through electrostatic, hydrophilic and H-bonding contacts with overall binding constants of KCh-15-tRNA=4.1 (±0.60)×10(3)M(-1), KCh-100-tRNA=5.7 (±0.8)×10(3)M(-1) and KCh-200-tRNA=1.2 (±0.3)×10(4)M(-1). As chitosan size increases more stable polymer-tRNA conjugate is formed. AFM images showed major tRNA aggregation and particle formation occurred as chitosan concentration increased. Even though chitosan induced major biopolymer structural changes, tRNA remains in A-family structure. Copyright © 2015 Elsevier B.V. All rights reserved.

Enumeration of RNA complexes via random matrix theory.

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Andersen, Jørgen E; Chekhov, Leonid O; Penner, Robert C; Reidys, Christian M; Sułkowski, Piotr

2013-04-01

In the present article, we review a derivation of the numbers of RNA complexes of an arbitrary topology. These numbers are encoded in the free energy of the Hermitian matrix model with potential V(x)=x2/2-stx/(1-tx), where s and t are respective generating parameters for the number of RNA molecules and hydrogen bonds in a given complex. The free energies of this matrix model are computed using the so-called topological recursion, which is a powerful new formalism arising from random matrix theory. These numbers of RNA complexes also have profound meaning in mathematics: they provide the number of chord diagrams of fixed genus with specified numbers of backbones and chords as well as the number of cells in Riemann's moduli spaces for bordered surfaces of fixed topological type.

Uganda's National Transmission Backbone Infrastructure Project: Technical Challenges and the Way Forward

Science.gov (United States)

Bulega, T.; Kyeyune, A.; Onek, P.; Sseguya, R.; Mbabazi, D.; Katwiremu, E.

2011-10-01

Several publications have identified technical challenges facing Uganda's National Transmission Backbone Infrastructure project. This research addresses the technical limitations of the National Transmission Backbone Infrastructure project, evaluates the goals of the project, and compares the results against the technical capability of the backbone. The findings of the study indicate a bandwidth deficit, which will be addressed by using dense wave division multiplexing repeaters, leasing bandwidth from private companies. Microwave links for redundancy, a Network Operation Center for operation and maintenance, and deployment of wireless interoperability for microwave access as a last-mile solution are also suggested.

31P T2s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the human brain at 7T

Science.gov (United States)

Klomp, Dennis W.J.; Wijnen, Jannie P.

2017-01-01

Purpose To determine the phosphorus‐31 T2s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the healthy human brain at 7T. Methods A 3D chemical shift imaging multi‐echo sequence with composite block pulses for refocusing was used to measure one free induction decay (FID) and seven full echoes with an echo spacing of 45 ms on the brain of nine healthy volunteers (age range 22–45 years; average age 27 ± 8 years). Spectral fitting was used to determine the change in metabolic signal amplitude with echo time. Results The average apparent T2s with their standard deviation were 202 ± 6 ms, 129 ± 6 ms, 86 ± 2 ms, 214 ± 10 ms, and 213 ± 11 ms for phosphoethanolamine, phosphocholine, inorganic phosphate, glycerophosphoethanolamine, and glycerophosphocholine, respectively. Conclusion The determined apparent T2 for phosphoethanolamine, glycerophosphocholine, and glycerophosphoethanolamine is approximately 200 ms. The lower apparent T2 value for phosphocholine is attributed to the overlap of this resonance with the 3‐phosphorous resonance of 2,3‐diphosphoglycerate from blood, with an apparent shorter T2. Omitting the FID signal and the first echo of phosphocholine leads to a T2 of 182 ± 7 ms, whereas a biexponential analysis leads to 203 ± 4 ms. These values are more in line with phosphoethanolamine and the phosphodiesters. The short T2 of inorganic phosphate is subscribed to the fast reversible exchange with γ‐adenosine triphosphate, which is mediated by glyceraldehyde‐3‐phosphate dehydrogenase and phosphoglycerate kinase within the glycolytic pathway. Magn Reson Med 80:29–35, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any

Some fractal properties of the percolating backbone in two dimensions

International Nuclear Information System (INIS)

Laidlaw, D.; MacKay, G.; Jan, N.

1987-01-01

A new algorithm is presented, based on elements of artificial intelligence theory, to determine the fractal properties of the backbone of the incipient infinite cluster. It is found that fractal dimensionality of the backbone is d/sub f//sup BB/ = 1.61 +/- 0.01, the chemical dimensionality is d/sub t/ = 1.40 +/- 0.01, and the fractal dimension of the minimum path d/sub min/ = 1.15 +/- 0.02 for the two-dimensional triangular lattice

Lactobacillus plantarum possesses the capability for wall teichoic acid backbone alditol switching

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Bron Peter A

2012-09-01

Full Text Available Abstract Background Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. Results Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type. Conclusions The study presented here correlates structural differences in WTA to their functional characteristics, thereby

Smart Inulin-Based Polycationic Nanodevices for siRNA Delivery.

Science.gov (United States)

Cavallaro, G; Sardo, C; Scialabba, C; Licciardi, M; Giammona, G

2017-01-01

The advances of short interfering RNA (siRNA) mediated therapy provide a powerful option for the treatment of many diseases by silencing the expression of targeted genes including cancer development and progression. Inulin is a very simple and biocompatible polysaccharide proposed by our groups to produce interesting delivery systems for Nucleic Acid Based Drugs (NABDs), such as siRNA, either as polycations able to give polyplexes and polymeric coatings for nanosystems having a metallic core. In this research field, different functionalizing groups were linked to the inulin backbone with specific aims including oligoamine such as Ethylendiammine (EDA), Diethylediamine (DETA), Spermine, (SPM) etc. In this contribution the main Inulin-based nanodevices for the delivery of siRNA have been reported, analysed and compared with particular reference to their chemical design and structure, biocompatibility, siRNA complexing ability, silencing ability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements

International Nuclear Information System (INIS)

Sakamoto, Koichi; Kamiya, Masakatsu; Uchida, Takeshi; Kawano, Keiichi; Ishimori, Koichiro

2010-01-01

Research highlights: → The dynamic parameters for the backbone dynamics in Cyt c were determined. → The backbone mobility of Cyt c is highly restricted due to the covalently bound heme. → The backbone mobility of Cyt c is more restricted upon the oxidation of the heme. → The redox-dependent dynamics are shown in the backbone of Cyt c. → The backbone dynamics of Cyt c would regulate the electron transfer from Cyt c. -- Abstract: Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D 15 N NMR relaxation experiments. 15 N T 1 and T 2 values and 1 H- 15 N NOEs of uniformly 15 N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S 2 ), the effective correlation time for internal motion (τ e ), the 15 N exchange broadening contributions (R ex ) for each residue, and the overall correlation time (τ m ) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S 2 value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S 2 values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.

Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

International Nuclear Information System (INIS)

Giesen, Alexander W.; Homans, Steve W.; Brown, Jonathan Miles

2003-01-01

We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N i -H N i , N i -C' i-1 , H N i - C' i-1 ) in two tensor frames and only backbone H N -H N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 A with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms

Resolution of RNA using high-performance liquid chromatography

NARCIS (Netherlands)

Mclaughlin, L.W.; Bischoff, Rainer

1987-01-01

High-performance liquid chromatographic techniques can be very effective for the resolution and isolation of nucleic acids. The characteristic ionic (phosphodiesters) and hydrophobic (nucleobases) properties of RNAs can be exploited for their separation. In this respect anion-exchange and

MCBT: Multi-Hop Cluster Based Stable Backbone Trees for Data Collection and Dissemination in WSNs

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Tae-Jin Lee

2009-07-01

Full Text Available We propose a stable backbone tree construction algorithm using multi-hop clusters for wireless sensor networks (WSNs. The hierarchical cluster structure has advantages in data fusion and aggregation. Energy consumption can be decreased by managing nodes with cluster heads. Backbone nodes, which are responsible for performing and managing multi-hop communication, can reduce the communication overhead such as control traffic and minimize the number of active nodes. Previous backbone construction algorithms, such as Hierarchical Cluster-based Data Dissemination (HCDD and Multicluster, Mobile, Multimedia radio network (MMM, consume energy quickly. They are designed without regard to appropriate factors such as residual energy and degree (the number of connections or edges to other nodes of a node for WSNs. Thus, the network is quickly disconnected or has to reconstruct a backbone. We propose a distributed algorithm to create a stable backbone by selecting the nodes with higher energy or degree as the cluster heads. This increases the overall network lifetime. Moreover, the proposed method balances energy consumption by distributing the traffic load among nodes around the cluster head. In the simulation, the proposed scheme outperforms previous clustering schemes in terms of the average and the standard deviation of residual energy or degree of backbone nodes, the average residual energy of backbone nodes after disseminating the sensed data, and the network lifetime.

Versatile phosphite ligands based on silsesquioxane backbones

NARCIS (Netherlands)

van der Vlugt, JI; Ackerstaff, J; Dijkstra, TW; Mills, AM; Kooijman, H; Spek, AL; Meetsma, A; Abbenhuis, HCL; Vogt, D

Silsesquioxanes are employed as ligand backbones for the synthesis of novel phosphite compounds with 3,3'-5,5'-tetrakis(tert-butyl)-2,2'-di-oxa-1,1'-biphenyl substituents. Both mono- and bidentate phosphites are prepared in good yields. Two types of silsesquioxanes are employed as starting

Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs.

Science.gov (United States)

Mutisya, Daniel; Selvam, Chelliah; Lunstad, Benjamin D; Pallan, Pradeep S; Haas, Amanda; Leake, Devin; Egli, Martin; Rozners, Eriks

2014-06-01

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3'-CH2-CO-NH-5' amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5'-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pyrite footprinting of RNA

International Nuclear Information System (INIS)

Schlatterer, Jörg C.; Wieder, Matthew S.; Jones, Christopher D.; Pollack, Lois; Brenowitz, Michael

2012-01-01

Highlights: ► RNA structure is mapped by pyrite mediated · OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High · OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. -- Abstract: In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ( · OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS 2 ) can produce sufficient · OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to · OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated · OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

Probing adenine rings and backbone linkages using base specific isotope-edited Raman spectroscopy: application to group II intron ribozyme domain V.

Science.gov (United States)

Chen, Yuanyuan; Eldho, Nadukkudy V; Dayie, T Kwaku; Carey, Paul R

2010-04-27

Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, "D5", which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all of its adenine residues labeled with (13)C and (15)N and utilizing Raman difference spectroscopy, we identify the conformationally sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg(2+) binding was explored by analyzing the Raman difference spectra for [D5 + Mg(2+)] minus [D5 no Mg(2+)], for D5 unlabeled, or D5 labeled with (13)C/(15)N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg(2+) binding at the N7 position. In the A-labeled spectra we attribute a Raman differential near 1450 cm(-1) and changes of intensity at 1296 cm(-1) to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg(2+) binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm(-1) upon magnesium binding is due to a "tightening up" (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine-labeled D5, small changes in the adenine backbone bond signatures in the 810-830 cm(-1) region suggest that small conformational changes occur in the tetraloop and bulge regions upon binding of Mg(2+). The PO(2)(-) stretching vibration, near 1100 cm(-1), from the nonbridging phosphate groups, probes the effect of Mg(2+)-hydrate inner-sphere interactions that cause an upshift. In turn, the upshift is modulated by the presence of monovalent cations since in the presence of Na(+) and Li(+) the upshift is 23 +/- 2 cm(-1) while in the presence of K(+) and Cs(+) it is 13 +/- 3 cm(-1), a finding that correlates

Integrative technology of massage manipulations in physical rehabilitation of students with backbone pathology

Directory of Open Access Journals (Sweden)

V.I. Kotelevskiy

2016-06-01

Full Text Available Purpose:to analyze effectiveness of massage manipulations’ integrative technology in physical rehabilitation of higher educational establishments’ students with backbone pathology. Material: in the research 195 students of 19-20 years’ age participated. All students had periodical initial neurological symptoms of functional pathology and first stage osteochondrosis in different parts of backbone. We conducted a course of 10 sessions of therapeutic massage. Results: the sense of massage integrative technology is that every specialist shall have certain optimal set of skills and knowledge in technique of manipulation sessions of massage. Integrative technology of massage manipulations consists of psycho-corrective and manipulation parts. It considers psycho-somatic, mechanical and reflex rehabilitation aspects of patho-genesis of backbone functional disorders and vertebral osteochondrosis. Conclusions: depending on pathological process or backbone functional state of every person (peculiarities of his (her psycho-somatic status or, even, his (her bents. Individual approach in choice of strategy, tactic and methodological provisioning of massage session shall be used.

High-sensitivity detection of polysaccharide using phosphodiesters quaternary ammonium salt as probe by decreased resonance light scattering.

Science.gov (United States)

Chen, Zhanguang; Liu, Guoliang; Chen, Maohuai; Wu, Mingyao

2009-07-15

Phosphodiesters quaternary ammonium salt (PQAS) displayed quite intense light scattering in aqueous solution under the optimum condition. In addition, the resonance light scattering (RLS) signal of PQAS was remarkably decreased after adding trace amount polysaccharide with the maximum peak located at 391 nm. It was found that the decreased RLS intensity of the PQAS-PPGL system (DeltaI(RLS)) was in proportion to PPGL concentration in the range of 0.1-30 ng mL(-1), with a lower detection limit of 0.05 ng mL(-1). Based on this rare decreased RLS phenomenon, the novel method of the determination of purified polysaccharide of Gracilaria Lemaneiformis (PPGL) at nanogram level was proposed in this contribution. The proposed approach was used to determine purified polysaccharide extracted from Gracilaria Lemaneiformis with satisfactory results. Compared with the reported polysaccharide assays, this proposed method has good selectivity, high sensitivity and is especially simple and convenient. Moreover, the mechanism of the reaction between PQAS and polysaccharide was investigated by RLS, fluorescence, and fluorescence lifetime spectra.

Selective backbone labelling of ILV methyl labelled proteins

International Nuclear Information System (INIS)

Sibille, Nathalie; Hanoulle, Xavier; Bonachera, Fanny; Verdegem, Dries; Landrieu, Isabelle; Wieruszeski, Jean-Michel; Lippens, Guy

2009-01-01

Adding the 13 C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of 12 C labelled glucose instead of the commonly used ( 2 D, 13 C) glucose leads not only to the 13 C labelling of (I, L, V) methyls but also to the selective 13 C labelling of the backbone C α and CO carbons of the Ile and Val residues. As a result, the backbone ( 1 H, 15 N) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue NOE effect, and should therefore be applicable to larger systems

Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid.

Science.gov (United States)

Babbitt, Gregory A; Alawad, Mohammed A; Schulze, Katharina V; Hudson, André O

2014-01-01

While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an 'accessory' during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

(α,α-dimethyl)glycyl (dmg) PNAs: achiral PNA analogs that form stronger hybrids with cDNA relative to isosequential RNA.

Science.gov (United States)

Gourishankar, Aland; Ganesh, Krishna N

2012-01-01

The design and facile synthesis of sterically constrained new analogs of PNA having gem-dimethyl substitutions on glycine (dmg-PNA-T) is presented. The PNA oligomers [aminoethyl dimethylglycyl (aedmg) and aminopropyl dimethylglycyl (apdmg)] synthesized from the monomers 6 and 12) effected remarkable stabilization of homothyminePNA(2):homoadenine DNA/RNA triplexes and mixed base sequence duplexes with target cDNA or RNA. They show a higher binding to DNA relative to that with isosequential RNA. This may be a structural consequence of the sterically rigid gem-dimethyl group, imposing a pre-organized conformation favorable for complex formation with cDNA. The results complement our previous work that had demonstrated that cyclohexanyl-PNAs favor binding with cRNA compared with cDNA and imply that the biophysical and structural properties of PNAs can be directed by introduction of the right rigidity in PNA backbone devoid of chirality. This approach of tweaking selectivity in binding of PNA constructs by installing gem-dimethyl substitution in PNA backbone can be extended to further fine-tuning by similar substitution in the aminoethyl segment as well either individually or in conjunction with present substitution.

Peptoid-Peptide hybrid backbone architectures

DEFF Research Database (Denmark)

Olsen, Christian Adam

2010-01-01

Peptidomimetic oligomers and foldamers have received considerable attention for over a decade, with beta-peptides and the so-called peptoids (N-alkylglycine oligomers) representing prominent examples of such architectures. Lately, hybrid or mixed backbones consisting of both alpha- and beta......-amino acids (alpha/beta-peptides) have been investigated in some detail as well. The present Minireview is a survey of the literature concerning hybrid structures of alpha-amino acids and peptoids, including beta-peptoids (N-alkyl-beta-alanine oligomers), and is intended to give an overview of this area...

Analisa Perbandingan Quality Of Service (QoS) pada Jaringan Backbone Non-MPLS dengan Jaringan Backbone MPLS Menggunakan Routing Protocol OSPF di PT. Telekomunikasi Indonesia, Tbk. Witel Ridar Riau

OpenAIRE

Silaban, Nestor Hasudungan; Sari, Linna Oktaviana; Anhar, Anhar

2015-01-01

The development of telecommunications technology based on Internet Protocol (IP) is now growing with the competitiveness of the telecommunications company to improve the quality of service to consumers. It can be obtained by increasing the quality backbone network using Multi Protocol Label Switching (MPLS). MPLS is a new technology to forward the packet to the backbone network without changing the existing network structure. The main idea is to construct a replacement MPLS paths using label ...

Five Principles of Industrialized Transformation for Successfully Building an Operational Backbone

DEFF Research Database (Denmark)

Winkler, Till J.; Kettunen, Petteri

2018-01-01

approach that is underpinned by five principles—template-based, business-driven, matrix-organized, tight supplier steering and cascaded planning. The UPM case provides important lessons for transformation leaders seeking to build, expand or develop a value-adding operational backbone.......To move into the digital age, a globally operating company needs to have in place an operational backbone, but many struggle with achieving this and the associated transformation program. Based on the experience of UPM, a Finnish forest industry company, we describe an industrialized transformation...

AbDesign: An algorithm for combinatorial backbone design guided by natural conformations and sequences.

Science.gov (United States)

Lapidoth, Gideon D; Baran, Dror; Pszolla, Gabriele M; Norn, Christoffer; Alon, Assaf; Tyka, Michael D; Fleishman, Sarel J

2015-08-01

Computational design of protein function has made substantial progress, generating new enzymes, binders, inhibitors, and nanomaterials not previously seen in nature. However, the ability to design new protein backbones for function--essential to exert control over all polypeptide degrees of freedom--remains a critical challenge. Most previous attempts to design new backbones computed the mainchain from scratch. Here, instead, we describe a combinatorial backbone and sequence optimization algorithm called AbDesign, which leverages the large number of sequences and experimentally determined molecular structures of antibodies to construct new antibody models, dock them against target surfaces and optimize their sequence and backbone conformation for high stability and binding affinity. We used the algorithm to produce antibody designs that target the same molecular surfaces as nine natural, high-affinity antibodies; in five cases interface sequence identity is above 30%, and in four of those the backbone conformation at the core of the antibody binding surface is within 1 Å root-mean square deviation from the natural antibodies. Designs recapitulate polar interaction networks observed in natural complexes, and amino acid sidechain rigidity at the designed binding surface, which is likely important for affinity and specificity, is high compared to previous design studies. In designed anti-lysozyme antibodies, complementarity-determining regions (CDRs) at the periphery of the interface, such as L1 and H2, show greater backbone conformation diversity than the CDRs at the core of the interface, and increase the binding surface area compared to the natural antibody, potentially enhancing affinity and specificity. © 2015 Wiley Periodicals, Inc.

Structural test of the parameterized-backbone method for protein design.

Science.gov (United States)

Plecs, Joseph J; Harbury, Pehr B; Kim, Peter S; Alber, Tom

2004-09-03

Designing new protein folds requires a method for simultaneously optimizing the conformation of the backbone and the side-chains. One approach to this problem is the use of a parameterized backbone, which allows the systematic exploration of families of structures. We report the crystal structure of RH3, a right-handed, three-helix coiled coil that was designed using a parameterized backbone and detailed modeling of core packing. This crystal structure was determined using another rationally designed feature, a metal-binding site that permitted experimental phasing of the X-ray data. RH3 adopted the intended fold, which has not been observed previously in biological proteins. Unanticipated structural asymmetry in the trimer was a principal source of variation within the RH3 structure. The sequence of RH3 differs from that of a previously characterized right-handed tetramer, RH4, at only one position in each 11 amino acid sequence repeat. This close similarity indicates that the design method is sensitive to the core packing interactions that specify the protein structure. Comparison of the structures of RH3 and RH4 indicates that both steric overlap and cavity formation provide strong driving forces for oligomer specificity.

Directional virtual backbone based data aggregation scheme for Wireless Visual Sensor Networks.

Science.gov (United States)

Zhang, Jing; Liu, Shi-Jian; Tsai, Pei-Wei; Zou, Fu-Min; Ji, Xiao-Rong

2018-01-01

Data gathering is a fundamental task in Wireless Visual Sensor Networks (WVSNs). Features of directional antennas and the visual data make WVSNs more complex than the conventional Wireless Sensor Network (WSN). The virtual backbone is a technique, which is capable of constructing clusters. The version associating with the aggregation operation is also referred to as the virtual backbone tree. In most of the existing literature, the main focus is on the efficiency brought by the construction of clusters that the existing methods neglect local-balance problems in general. To fill up this gap, Directional Virtual Backbone based Data Aggregation Scheme (DVBDAS) for the WVSNs is proposed in this paper. In addition, a measurement called the energy consumption density is proposed for evaluating the adequacy of results in the cluster-based construction problems. Moreover, the directional virtual backbone construction scheme is proposed by considering the local-balanced factor. Furthermore, the associated network coding mechanism is utilized to construct DVBDAS. Finally, both the theoretical analysis of the proposed DVBDAS and the simulations are given for evaluating the performance. The experimental results prove that the proposed DVBDAS achieves higher performance in terms of both the energy preservation and the network lifetime extension than the existing methods.

Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

International Nuclear Information System (INIS)

Lopez del Amo, Juan-Miguel; Fink, Uwe; Reif, Bernd

2010-01-01

We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual 15 N-T 1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s -1 . Backbone amide 15 N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D 2 O is employed as a solvent for sample preparation. Due to the intrinsically long 15 N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

Science.gov (United States)

Sharma, Rakhi; Sahu, Bhubanananda; Ray, Malay K; Deshmukh, Mandar V

2015-04-01

Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W.

Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

Science.gov (United States)

Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

2016-01-01

Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

Induced helical backbone conformations of self-organizable dendronized polymers.

Science.gov (United States)

Rudick, Jonathan G; Percec, Virgil

2008-12-01

Control of function through the primary structure of a molecule presents a significant challenge with valuable rewards for nanoscience. Dendritic building blocks encoded with information that defines their three-dimensional shape (e.g., flat-tapered or conical) and how they associate with each other are referred to as self-assembling dendrons. Self-organizable dendronized polymers possess a flat-tapered or conical self-assembling dendritic side chain on each repeat unit of a linear polymer backbone. When appended to a covalent polymer, the self-assembling dendrons direct a folding process (i.e., intramolecular self-assembly). Alternatively, intermolecular self-assembly of dendrons mediated by noncovalent interactions between apex groups can generate a supramolecular polymer backbone. Self-organization, as we refer to it, is the spontaneous formation of periodic and quasiperiodic arrays from supramolecular elements. Covalent and supramolecular polymers jacketed with self-assembling dendrons self-organize. The arrays are most often comprised of cylindrical or spherical objects. The shape of the object is determined by the primary structure of the dendronized polymer: the structure of the self-assembling dendron and the length of the polymer backbone. It is therefore possible to predictably generate building blocks for single-molecule nanotechnologies or arrays of supramolecules for bottom-up self-assembly. We exploit the self-organization of polymers jacketed with self-assembling dendrons to elucidate how primary structure determines the adopted conformation and fold (i.e., secondary and tertiary structure), how the supramolecules associate (i.e., quaternary structure), and their resulting functions. A combination of experimental techniques is employed to interrogate the primary, secondary, tertiary, and quaternary structure of the self-organizable dendronized polymers. We refer to the process by which we interpolate between the various levels of structural

Instant Backbone.js application development

CERN Document Server

Hunter, Thomas

2013-01-01

Get to grips with a new technology, understand what it is and what it can do for you, and then get to work with the most important features and tasks. This book is a practical, step-by-step tutorial that will teach you to build Backbone.js applications quickly and efficiently.This book is targeted towards developers. It is assumed that you have at least a basic understanding of JavaScript and jQuery selectors. If you are interested in building dynamic Single Page Applications that interact heavily with a backend server, then this is the book for you.

Backbone conformation affects duplex initiation and duplex propagation in hybridisation of synthetic H-bonding oligomers.

Science.gov (United States)

Iadevaia, Giulia; Núñez-Villanueva, Diego; Stross, Alexander E; Hunter, Christopher A

2018-06-06

Synthetic oligomers equipped with complementary H-bond donor and acceptor side chains form multiply H-bonded duplexes in organic solvents. Comparison of the duplex forming properties of four families of oligomers with different backbones shows that formation of an extended duplex with three or four inter-strand H-bonds is more challenging than formation of complexes that make only two H-bonds. The stabilities of 1 : 1 complexes formed between length complementary homo-oligomers equipped with either phosphine oxide or phenol recognition modules were measured in toluene. When the backbone is very flexible (pentane-1,5-diyl thioether), the stability increases uniformly by an order of magnitude for each additional base-pair added to the duplex: the effective molarities for formation of the first intramolecular H-bond (duplex initiation) and subsequent intramolecular H-bonds (duplex propagation) are similar. This flexible system is compared with three more rigid backbones that are isomeric combinations of an aromatic ring and methylene groups. One of the rigid systems behaves in exactly the same way as the flexible backbone, but the other two do not. For these systems, the effective molarity for formation of the first intramolecular H-bond is the same as that found for the other two backbones, but additional H-bonds are not formed between the longer oligomers. The effective molarities are too low for duplex propagation in these systems, because the oligomer backbones cannot adopt conformations compatible with formation of an extended duplex.

Study of muscular skeletal apparatus’s functional state of junior sportsmen-power lifters, who have backbone verterbral abnormalities

Directory of Open Access Journals (Sweden)

V.R. Ilmatov

2015-10-01

Full Text Available Purpose: determination of abnormalities and disorders of muscular skeletal apparatuses’ status of power lifters, who have vertebral abnormalities of backbone. Material: 58 junior sportsmen participated in the research. 36 sportsmen were the main group of the research and had vertebral disorders in backbone. For posture testing visual examination was used. Backbone mobility was tested with goniometry method. Flat feet were registered with plantography method. Results: we determined posture abnormalities in sagittal and frontal planes; feet flat, limited maximal movements in thoracic and lumbar spines. It was determined that the most limited were rotational movements and backbone unbending. The next were side bents. These limitations were accompanied by pain syndrome. These observations indirectly confirmed theory of direct interaction of backbone structures with nervous structures. It is also a confirmation of vertebral abnormalities’ presence in junior sportsmen. Conclusions: it was found that in junior sportsmen - power lifters with backbone pathologies in 100% of cases symptoms are determined by local limitations of backbone mobility with pain syndrome. In 35% of cases they are accompanied by posture’s disorders and feet flat. Orientation and methodic of rehabilitation of such sportsmen have been determined.

Performance of Flow-Aware Networking in LTE backbone

DEFF Research Database (Denmark)

Sniady, Aleksander; Soler, José

2012-01-01

technologies, such as Long Term Evolution (LTE). This paper proposes usage of a modified Flow Aware Networking (FAN) technique for enhancing Quality of Service (QoS) in the all-IP transport networks underlying LTE backbone. The results obtained with OPNET Modeler show that FAN, in spite of being relatively...

Energies and 2 '-Hydroxyl Group Orientations of RNA Backbone Conformations. Benchmark CCSD(T)/CBS Database, Electronic Analysis, and Assessment of DFT Methods and MD Simulations

Czech Academy of Sciences Publication Activity Database

Mládek, Arnošt; Banáš, P.; Jurečka, P.; Otyepka, M.; Zgarbová, M.; Šponer, Jiří

2014-01-01

Roč. 10, č. 1 (2014), s. 463-480 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GAP208/12/1878; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : DENSITY-FUNCTIONAL THEORY * SUGAR-PHOSPHATE BACKBONE * QUANTUM-CHEMICAL CALCULATIONS Subject RIV: BO - Biophysics Impact factor: 5.498, year: 2014

Towards a natural classification and backbone tree for Sordariomycete

Digital Repository Service at National Institute of Oceanography (India)

Maharachchikumbura, S.S.N.; Hyde, K.D.; Jones, E.B.G.; McKenzie, E.H.C.; Huang, S.-K.; Abdel-Wahab, M.A.; Daranagama, D.A.; Dayarathne, M.; D'souza, M.J.; Goonasekara, I.D.; Hongsanan, S.; Jayawardena, R.S.; Kirk, P.M.; Konta, S.; Liu, J.-K.; Liu, Z.-Y.; Norphanphoun, C.; Pang, K.-L.; Perera, R.H.; Senanayake, I.C.; Shang, Q.; Shenoy, B.D.; Xiao, Y.; Bahkali, A.H.; Kang, J.; Somrothipol, S.; Suetrong, S.; Wen, T.; Xu, J.

, lichenized or lichenicolous taxa The class includes freshwater, marine and terrestrial taxa and has a worldwide distribution This paper provides an updated outline of the Sordariomycetes and a backbone tree incorporating asexual and sexual genera in the class...

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

Science.gov (United States)

Sharp, Kim A; O'Brien, Evan; Kasinath, Vignesh; Wand, A Joshua

2015-05-01

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH  entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. © 2015 Wiley Periodicals, Inc.

Optical alignment control of polyimide molecules containing azobenzene in the backbone structure

International Nuclear Information System (INIS)

Sakamoto, Kenji; Usami, Kiyoaki; Sasaki, Toru; Kanayama, Takashi; Ushioda, Sukekatsu

2004-01-01

Using polarized infrared absorption spectroscopy, we have determined the orientation of the polyimide backbone structure in photo-alignment films for liquid crystals (LC). The polyimide used in this study contains azobenzene in the backbone structure. Photo-alignment treatment was performed on the corresponding polyamic acid film, using a light source of wavelength 340-500 nm. The polyamic acid film (∼16 nm thick) was first irradiated at normal incidence with linearly polarized light (LP-light) of 156 J/cm 2 , and then oblique angle irradiation of unpolarized light (UP-light) was performed in the plane of incidence perpendicular to the polarization direction of the LP-light. The UP-light exposure was varied up to 882 J/cm 2 . We found that the average inclination angle of the polyimide backbone structure, measured from the surface plane, increases almost linearly with UP-light exposure. On the other hand, the in-plane anisotropy induced by the first irradiation with LP-light decreases with the increase of UP-light exposure

Analysis of stationary availability factor of two-level backbone computer networks with arbitrary topology

Science.gov (United States)

Rahman, P. A.

2018-05-01

This scientific paper deals with the two-level backbone computer networks with arbitrary topology. A specialized method, offered by the author for calculation of the stationary availability factor of the two-level backbone computer networks, based on the Markov reliability models for the set of the independent repairable elements with the given failure and repair rates and the methods of the discrete mathematics, is also discussed. A specialized algorithm, offered by the author for analysis of the network connectivity, taking into account different kinds of the network equipment failures, is also observed. Finally, this paper presents an example of calculation of the stationary availability factor for the backbone computer network with the given topology.

5D {sup 13}C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

Energy Technology Data Exchange (ETDEWEB)

Novacek, Jiri [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic); Zawadzka-Kazimierczuk, Anna [University of Warsaw, Faculty of Chemistry (Poland); Papouskova, Veronika; Zidek, Lukas, E-mail: lzidek@chemi.muni.cz [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic); Sanderova, Hana; Krasny, Libor [Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology (Czech Republic); Kozminski, Wiktor [University of Warsaw, Faculty of Chemistry (Poland); Sklenar, Vladimir [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic)

2011-05-15

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of {sup 13}C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

Automated backbone assignment of labeled proteins using the threshold accepting algorithm

International Nuclear Information System (INIS)

Leutner, Michael; Gschwind, Ruth M.; Liermann, Jens; Schwarz, Christian; Gemmecker, Gerd; Kessler, Horst

1998-01-01

The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C β information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A 2 , a protein with 124 residues

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

International Nuclear Information System (INIS)

Hrovat, Andrea; Bluemel, Markus; Loehr, Frank; Mayhew, Stephen G.; Rueterjans, Heinz

1997-01-01

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15 N- 1 H correlation NMR spectroscopy.T 1 , T 2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state

Ebolavirus VP35 uses a bimodal strategy to bind dsRNA for innate immune suppression

Energy Technology Data Exchange (ETDEWEB)

Kimberlin, Christopher R.; Bornholdt, Zachary A.; Li, Sheng; Woods, Jr., Virgil L.; MacRae, Ian J.; Saphire, Erica Ollmann (Scripps); (UCSD)

2010-03-12

Ebolavirus causes a severe hemorrhagic fever and is divided into five distinct species, of which Reston ebolavirus is uniquely nonpathogenic to humans. Disease caused by ebolavirus is marked by early immunosuppression of innate immune signaling events, involving silencing and sequestration of double-stranded RNA (dsRNA) by the viral protein VP35. Here we present unbound and dsRNA-bound crystal structures of the dsRNA-binding domain of Reston ebolavirus VP35. The structures show that VP35 forms an unusual, asymmetric dimer on dsRNA binding, with each of the monomers binding dsRNA in a different way: one binds the backbone whereas the other caps the terminus. Additional SAXS, DXMS, and dsRNA-binding experiments presented here support a model of cooperative dsRNA recognition in which binding of the first monomer assists binding of the next monomer of the oligomeric VP35 protein. This work illustrates how ebolavirus VP35 could mask key recognition sites of molecules such as RIG-I, MDA-5, and Dicer to silence viral dsRNA in infection.

Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life

DEFF Research Database (Denmark)

Karst, Soeren M; Dueholm, Morten S; McIlroy, Simon J

2016-01-01

Ribosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies...... (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human...... gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity...

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

Science.gov (United States)

Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Generation of RNA in abiotic conditions.

Science.gov (United States)

di Mauro, Ernesto

Generation of RNA in abiotic conditions. Ernesto Di Mauro Dipartimento di Genetica Bi-ologia Molecolare, Universit` "Sapienza" Roma, Italy. a At least four conditions must be satisfied for the spontaneous generation of (pre)-genetic poly-mers: 1) availability of precursors that are activated enough to spontaneously polymerize. Preliminary studies showed that (a) nucleic bases and acyclonucleosides can be synthesized from formamide H2NCOH by simply heating with prebiotically available mineral catalysts [last reviewed in (1)], and that b) nucleic bases can be phosphorylated in every possible posi-tion [2'; 3'; 5'; cyclic 2',3'; cyclic 3',5' (2)]. The higher stability of the cyclic forms allows their accumulation. 2) A polymerization mechanism. A reaction showing the formation of RNA polymers starting from prebiotically plausible precursors (3',5' cyclic GMP and 3', 5'cyclic AMP) was recently reported (3). Polymerization in these conditions is thermodynamically up-hill and an equilibrium is attained that limits the maximum length of the polymer produced to about 40 nucleotides for polyG and 100 nucleotides for polyA. 3) Ligation of the synthesized oligomers. If this type of reaction could occur according to a terminal-joining mechanism and could generate canonical 3',5' phosphodiester bonds, exponential growth would be obtained of the generated oligomers. This type of reaction has been reported (4) , limited to homogeneous polyA sequences and leading to the production of polyA dimers and tetramers. What is still missing are: 4) mechanisms that provide the proof of principle for the generation of sequence complexity. We will show evidence for two mechanisms providing this proof of principle for simple complementary sequences. Namely: abiotic sequence complementary-driven terminal ligation and sequence-complementary terminal growth. In conclusion: all the steps leading to the generation of RNA in abiotic conditions are satisfied. (1) R Saladino, C Crestini, F

Primordial soup or vinaigrette: did the RNA world evolve at acidic pH?

Directory of Open Access Journals (Sweden)

Bernhardt Harold S

2012-01-01

Full Text Available Abstract Background The RNA world concept has wide, though certainly not unanimous, support within the origin-of-life scientific community. One view is that life may have emerged as early as the Hadean Eon 4.3-3.8 billion years ago with an atmosphere of high CO2 producing an acidic ocean of the order of pH 3.5-6. Compatible with this scenario is the intriguing proposal that life arose within alkaline (pH 9-11 deep-sea hydrothermal vents like those of the 'Lost City', with the interface with the acidic ocean creating a proton gradient sufficient to drive the first metabolism. However, RNA is most stable at pH 4-5 and is unstable at alkaline pH, raising the possibility that RNA may have first arisen in the acidic ocean itself (possibly near an acidic hydrothermal vent, acidic volcanic lake or comet pond. As the Hadean Eon progressed, the ocean pH is inferred to have gradually risen to near neutral as atmospheric CO2 levels decreased. Presentation of the hypothesis We propose that RNA is well suited for a world evolving at acidic pH. This is supported by the enhanced stability at acidic pH of not only the RNA phosphodiester bond but also of the aminoacyl-(tRNA and peptide bonds. Examples of in vitro-selected ribozymes with activities at acid pH have recently been documented. The subsequent transition to a DNA genome could have been partly driven by the gradual rise in ocean pH, since DNA has greater stability than RNA at alkaline pH, but not at acidic pH. Testing the hypothesis We have proposed mechanisms for two key RNA world activities that are compatible with an acidic milieu: (i non-enzymatic RNA replication of a hemi-protonated cytosine-rich oligonucleotide, and (ii specific aminoacylation of tRNA/hairpins through triple helix interactions between the helical aminoacyl stem and a single-stranded aminoacylating ribozyme. Implications of the hypothesis Our hypothesis casts doubt on the hypothesis that RNA evolved in the vicinity of alkaline

Turning limited experimental information into 3D models of RNA.

Science.gov (United States)

Flores, Samuel Coulbourn; Altman, Russ B

2010-09-01

Our understanding of RNA functions in the cell is evolving rapidly. As for proteins, the detailed three-dimensional (3D) structure of RNA is often key to understanding its function. Although crystallography and nuclear magnetic resonance (NMR) can determine the atomic coordinates of some RNA structures, many 3D structures present technical challenges that make these methods difficult to apply. The great flexibility of RNA, its charged backbone, dearth of specific surface features, and propensity for kinetic traps all conspire with its long folding time, to challenge in silico methods for physics-based folding. On the other hand, base-pairing interactions (either in runs to form helices or isolated tertiary contacts) and motifs are often available from relatively low-cost experiments or informatics analyses. We present RNABuilder, a novel code that uses internal coordinate mechanics to satisfy user-specified base pairing and steric forces under chemical constraints. The code recapitulates the topology and characteristic L-shape of tRNA and obtains an accurate noncrystallographic structure of the Tetrahymena ribozyme P4/P6 domain. The algorithm scales nearly linearly with molecule size, opening the door to the modeling of significantly larger structures.

Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector.

Science.gov (United States)

Miyafusa, Takamitsu; Shibuya, Risa; Honda, Shinya

2018-06-02

Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 3 10 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology. Copyright © 2018 Elsevier Inc. All rights reserved.

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

Science.gov (United States)

Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

2016-02-01

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

Constructing Battery-Aware Virtual Backbones in Wireless Sensor Networks

Directory of Open Access Journals (Sweden)

Yang Yuanyuan

2007-01-01

Full Text Available A critical issue in battery-powered sensor networks is to construct energy efficient virtual backbones for network routing. Recent study in battery technology reveals that batteries tend to discharge more power than needed and reimburse the over-discharged power if they are recovered. In this paper we first provide a mathematical battery model suitable for implementation in sensor networks. We then introduce the concept of battery-aware connected dominating set (BACDS and show that in general the minimum BACDS (MBACDS can achieve longer lifetime than the previous backbone structures. Then we show that finding a MBACDS is NP-hard and give a distributed approximation algorithm to construct the BACDS. The resulting BACDS constructed by our algorithm is at most opt size, where is the maximum node degree and opt is the size of an optimal BACDS. Simulation results show that the BACDS can save a significant amount of energy and achieve up to longer network lifetime than previous schemes.

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

Science.gov (United States)

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the

Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA

Science.gov (United States)

Smith, Rachel M.; Marshall, Jacqueline J. T.; Jacklin, Alistair J.; Retter, Susan E.; Halford, Stephen E.; Sobott, Frank

2013-01-01

Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A2B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A2B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A2B units, each bound to a recognition site, with two more A2B units bridging the complexes by protein–protein interactions between the nuclease domains. PMID:23147005

Promoter-wide hypermethylation of the ribosomal RNA gene promoter in the suicide brain.

Directory of Open Access Journals (Sweden)

Patrick O McGowan

Full Text Available BACKGROUND: Alterations in gene expression in the suicide brain have been reported and for several genes DNA methylation as an epigenetic regulator is thought to play a role. rRNA genes, that encode ribosomal RNA, are the backbone of the protein synthesis machinery and levels of rRNA gene promoter methylation determine rRNA transcription. METHODOLOGY/PRINCIPAL FINDINGS: We test here by sodium bisulfite mapping of the rRNA promoter and quantitative real-time PCR of rRNA expression the hypothesis that epigenetic differences in critical loci in the brain are involved in the pathophysiology of suicide. Suicide subjects in this study were selected for a history of early childhood neglect/abuse, which is associated with decreased hippocampal volume and cognitive impairments. rRNA was significantly hypermethylated throughout the promoter and 5' regulatory region in the brain of suicide subjects, consistent with reduced rRNA expression in the hippocampus. This difference in rRNA methylation was not evident in the cerebellum and occurred in the absence of genome-wide changes in methylation, as assessed by nearest neighbor. CONCLUSIONS/SIGNIFICANCE: This is the first study to show aberrant regulation of the protein synthesis machinery in the suicide brain. The data implicate the epigenetic modulation of rRNA in the pathophysiology of suicide.

Polyolefin backbone substitution in binders for low temperature powder injection moulding feedstocks.

Science.gov (United States)

Hausnerova, Berenika; Kuritka, Ivo; Bleyan, Davit

2014-02-27

This paper reports the substitution of polyolefin backbone binder components with low melting temperature carnauba wax for powder injection moulding applications. The effect of various binder compositions of Al₂O₃ feedstock on thermal degradation parameters is investigated by thermogravimetric analysis. Within the experimental framework 29 original feedstock compositions were prepared and the superiority of carnauba wax over the polyethylene binder backbone was demonstrated in compositions containing polyethylene glycol as the initial opening agent and governing the proper mechanism of the degradation process. Moreover, the replacement of synthetic polymer by the natural wax contributes to an increase of environmental sustainability of modern industrial technologies.

Internet Backbone in the Democratic Republic of Congo : Feasibility ...

International Development Research Centre (IDRC) Digital Library (Canada)

Internet Backbone in the Democratic Republic of Congo : Feasibility Study and Advocacy. During 7-10 February 2005, representatives of five francophone African countries (Cameroon, Morocco, Niger, Sénégal, and the Democratic Republic of Congo - DRC) met to consider ways and means of galvanizing the appropriation ...

Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

International Nuclear Information System (INIS)

Anesti, Anna-Maria; Simpson, Guy R; Price, Toby; Pandha, Hardev S; Coffin, Robert S

2010-01-01

Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF , we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials

Oxidation Responsive Polymers with a Triggered Degradation via Arylboronate Self-Immolative Motifs on a Polyphosphazene Backbone.

Science.gov (United States)

Iturmendi, Aitziber; Monkowius, Uwe; Teasdale, Ian

2017-02-21

Oxidation responsive polymers with triggered degradation pathways have been prepared via attachment of self-immolative moieties onto a hydrolytically unstable polyphosphazene backbone. After controlled main-chain growth, postpolymerization functionalization allows the preparation of hydrolytically stable poly(organo)phosphazenes decorated with a phenylboronic ester caging group. In oxidative environments, triggered cleavage of the caging group is followed by self-immolation, exposing the unstable glycine-substituted polyphosphazene which subsequently undergoes to backbone degradation to low-molecular weight molecules. As well as giving mechanistic insights, detailed GPC and 1 H and 31 P NMR analysis reveal the polymers to be stable in aqueous solutions, but show a selective, fast degradation upon exposure to hydrogen peroxide containing solutions. Since the post-polymerization functionalization route allows simple access to polymer backbones with a broad range of molecular weights, the approach of using the inorganic backbone as a platform significantly expands the toolbox of polymers capable of stimuli-responsive degradation.

Dynamic power control for wireless backbone mesh networks: a survey

CSIR Research Space (South Africa)

Olwal, TO

2010-01-01

Full Text Available points of failures, and robust against RF interference, obstacles or power outage. This is because WMRs forming wireless backbone mesh networks (WBMNs) are built on advanced physical technologies. Such nodes perform both accessing and forwarding...

Building the library of RNA 3D nucleotide conformations using the clustering approach

Directory of Open Access Journals (Sweden)

Zok Tomasz

2015-09-01

Full Text Available An increasing number of known RNA 3D structures contributes to the recognition of various RNA families and identification of their features. These tasks are based on an analysis of RNA conformations conducted at different levels of detail. On the other hand, the knowledge of native nucleotide conformations is crucial for structure prediction and understanding of RNA folding. However, this knowledge is stored in structural databases in a rather distributed form. Therefore, only automated methods for sampling the space of RNA structures can reveal plausible conformational representatives useful for further analysis. Here, we present a machine learning-based approach to inspect the dataset of RNA three-dimensional structures and to create a library of nucleotide conformers. A median neural gas algorithm is applied to cluster nucleotide structures upon their trigonometric description. The clustering procedure is two-stage: (i backbone- and (ii ribose-driven. We show the resulting library that contains RNA nucleotide representatives over the entire data, and we evaluate its quality by computing normal distribution measures and average RMSD between data points as well as the prototype within each cluster.

T. thermophila group I introns that cleave amide bonds

Science.gov (United States)

Joyce, Gerald F. (Inventor)

1997-01-01

The present invention relates to nucleic acid enzymes or enzymatic RNA molecules that are capable of cleaving a variety of bonds, including phosphodiester bonds and amide bonds, in a variety of substrates. Thus, the disclosed enzymatic RNA molecules are capable of functioning as nucleases and/or peptidases. The present invention also relates to compositions containing the disclosed enzymatic RNA molecule and to methods of making, selecting, and using such enzymes and compositions.

Green IGP Link Weights for Energy-efficiency and Load-balancing in IP Backbone Networks

OpenAIRE

Francois, Frederic; Wang, Ning; Moessner, Klaus; Georgoulas, Stylianos; Xu, Ke

2013-01-01

The energy consumption of backbone networks has become a primary concern for network operators and regulators due to the pervasive deployment of wired backbone networks to meet the requirements of bandwidth-hungry applications. While traditional optimization of IGP link weights has been used in IP based load-balancing operations, in this paper we introduce a novel link weight setting algorithm, the Green Load-balancing Algorithm (GLA), which is able to jointly optimize both energy efficiency ...

Structural Insights into RNA Recognition by the Alternate-Splicing Regulator CUG-Binding Protein 1

Energy Technology Data Exchange (ETDEWEB)

M Teplova; J Song; H Gaw; A Teplov; D Patel

2011-12-31

CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.

Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

International Nuclear Information System (INIS)

Garfinkel, S.; Thompson, J.A.; Cohen, R.B.; Brendler, T.; Safer, B.

1987-01-01

Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific 32 P-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect

Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

DEFF Research Database (Denmark)

Andersen, Christian Brix Folsted; Ballut, Lionel; Johansen, Jesper Sanderhoff

2006-01-01

exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides...... and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I....

Polyolefin Backbone Substitution in Binders for Low Temperature Powder Injection Moulding Feedstocks

Directory of Open Access Journals (Sweden)

Berenika Hausnerova

2014-02-01

Full Text Available This paper reports the substitution of polyolefin backbone binder components with low melting temperature carnauba wax for powder injection moulding applications. The effect of various binder compositions of Al2O3 feedstock on thermal degradation parameters is investigated by thermogravimetric analysis. Within the experimental framework 29 original feedstock compositions were prepared and the superiority of carnauba wax over the polyethylene binder backbone was demonstrated in compositions containing polyethylene glycol as the initial opening agent and governing the proper mechanism of the degradation process. Moreover, the replacement of synthetic polymer by the natural wax contributes to an increase of environmental sustainability of modern industrial technologies.

“Pinning strategy”: a novel approach for predicting the backbone ...

Indian Academy of Sciences (India)

Prakash

To assess the quality of the strategy, we define two measures. The first one ...... modular framework of the protein backbone; Protein Eng. 12. 1063–1073 .... Richardson J S, Getzoff E D and Richardson D C 1978 The beta bulge: a common ...

Impact of Backbone Fluorination on π-Conjugated Polymers in Organic Photovoltaic Devices: A Review

Directory of Open Access Journals (Sweden)

Nicolas Leclerc

2016-01-01

Full Text Available Solution-processed bulk heterojunction solar cells have experienced a remarkable acceleration in performances in the last two decades, reaching power conversion efficiencies above 10%. This impressive progress is the outcome of a simultaneous development of more advanced device architectures and of optimized semiconducting polymers. Several chemical approaches have been developed to fine-tune the optoelectronics and structural polymer parameters required to reach high efficiencies. Fluorination of the conjugated polymer backbone has appeared recently to be an especially promising approach for the development of efficient semiconducting polymers. As a matter of fact, most currently best-performing semiconducting polymers are using fluorine atoms in their conjugated backbone. In this review, we attempt to give an up-to-date overview of the latest results achieved on fluorinated polymers for solar cells and to highlight general polymer properties’ evolution trends related to the fluorination of their conjugated backbone.

Constructing Battery-Aware Virtual Backbones in Wireless Sensor Networks

Directory of Open Access Journals (Sweden)

Chi Ma

2007-05-01

Full Text Available A critical issue in battery-powered sensor networks is to construct energy efficient virtual backbones for network routing. Recent study in battery technology reveals that batteries tend to discharge more power than needed and reimburse the over-discharged power if they are recovered. In this paper we first provide a mathematical battery model suitable for implementation in sensor networks. We then introduce the concept of battery-aware connected dominating set (BACDS and show that in general the minimum BACDS (MBACDS can achieve longer lifetime than the previous backbone structures. Then we show that finding a MBACDS is NP-hard and give a distributed approximation algorithm to construct the BACDS. The resulting BACDS constructed by our algorithm is at most (8+Δopt size, where Δ is the maximum node degree and opt is the size of an optimal BACDS. Simulation results show that the BACDS can save a significant amount of energy and achieve up to 30% longer network lifetime than previous schemes.

Effect of backbone chemistry on hybridization thermodynamics of oligonucleic acids: a coarse-grained molecular dynamics simulation study.

Science.gov (United States)

Ghobadi, Ahmadreza F; Jayaraman, Arthi

2016-02-28

In this paper we study how varying oligonucleic acid backbone chemistry affects the hybridization/melting thermodynamics of oligonucleic acids. We first describe the coarse-grained (CG) model with tunable parameters that we developed to enable the study of both naturally occurring oligonucleic acids, such as DNA, and their chemically-modified analogues, such as peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). The DNA melting curves obtained using such a CG model and molecular dynamics simulations in an implicit solvent and with explicit ions match with the melting curves obtained using the empirical nearest-neighbor models. We use these CG simulations to then elucidate the effect of backbone flexibility, charge, and nucleobase spacing along the backbone on the melting curves, potential energy and conformational entropy change upon hybridization and base-pair hydrogen bond residence time. We find that increasing backbone flexibility decreases duplex thermal stability and melting temperature mainly due to increased conformational entropy loss upon hybridization. Removing charges from the backbone enhances duplex thermal stability due to the elimination of electrostatic repulsion and as a result a larger energetic gain upon hybridization. Lastly, increasing nucleobase spacing decreases duplex thermal stability due to decreasing stacking interactions that are important for duplex stability.

Future High Capacity Backbone Networks

DEFF Research Database (Denmark)

Wang, Jiayuan

are proposed. The work focuses on energy efficient routing algorithms in a dynamic optical core network environment, with Generalized MultiProtocol Label Switching (GMPLS) as the control plane. Energy ef- ficient routing algorithms for energy savings and CO2 savings are proposed, and their performance...... aiming for reducing the dynamic part of the energy consumption of the network may increase the fixed part of the energy consumption meanwhile. In the second half of the thesis, the conflict between energy efficiency and Quality of Service (QoS) is addressed by introducing a novel software defined......This thesis - Future High Capacity Backbone Networks - deals with the energy efficiency problems associated with the development of future optical networks. In the first half of the thesis, novel approaches for using multiple/single alternative energy sources for improving energy efficiency...

Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase.

Science.gov (United States)

Iqbal, Emil S; Dods, Kara K; Hartman, Matthew C T

2018-02-14

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic β-amino acids.

Smectic order and backbone anisotropy of a side-chain liquid crystalline polymer by Small-Angle Neutron Scattering

Science.gov (United States)

Noirez, L.; Pépy, G.; Keller, P.; Benguigui, L.

1991-07-01

We have simultaneously measured, for the first time, the extension of the polymer backbone of a side-chain liquid crystalline polymer and the intensity of the 001 Bragg reflection, which gives the smectic order parameter Psi as a function of temperature in the smectic phase. We have qualitatively demonstrated that the more the smectic phase is ordered, the more the polymer backbone is localized between the mesogenic layers. It is shown that the Landau theory allows us to relate the radius of gyration parallel to the magnetic field of the polymer backbone to the smectic order parameter. We also show that the Renz-Warner theory is suitable at low temperatures.

One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR).

Science.gov (United States)

Breunig, Christopher T; Durovic, Tamara; Neuner, Andrea M; Baumann, Valentin; Wiesbeck, Maximilian F; Köferle, Anna; Götz, Magdalena; Ninkovic, Jovica; Stricker, Stefan H

2018-01-01

Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR's widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo.

The snakelike chain character of unstructured RNA.

Science.gov (United States)

Jacobson, David R; McIntosh, Dustin B; Saleh, Omar A

2013-12-03

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod "wormlike chain" (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a "snakelike chain," characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Self-assembly of diphenylalanine backbone homologues and their combination with functionalized carbon nanotubes.

Science.gov (United States)

Dinesh, Bhimareddy; Squillaci, Marco A; Ménard-Moyon, Cécilia; Samorì, Paolo; Bianco, Alberto

2015-10-14

The integration of carbon nanotubes (CNTs) into organized nanostructures is of great interest for applications in materials science and biomedicine. In this work we studied the self-assembly of β and γ homologues of diphenylalanine peptides under different solvent and pH conditions. We aimed to investigate the role of peptide backbone in tuning the formation of different types of nanostructures alone or in combination with carbon nanotubes. In spite of having the same side chain, β and γ peptides formed distinctively different nanofibers, a clear indication of the role played by the backbone homologation on the self-assembly. The variation of the pH allowed to transform the nanofibers into spherical structures. Moreover, the co-assembly of β and γ peptides with carbon nanotubes covalently functionalized with the same peptide generated unique dendritic assemblies. This comparative study on self-assembly using diphenylalanine backbone homologues and of the co-assembly with CNT covalent conjugates is the first example exploring the capacity of β and γ peptides to adopt precise nanostructures, particularly in combination with carbon nanotubes. The dendritic organization obtained by mixing carbon nanotubes and peptides might find interesting applications in tissue engineering and neuronal interfacing.

Performance Analysis of Space Information Networks with Backbone Satellite Relaying for Vehicular Networks

Directory of Open Access Journals (Sweden)

Jian Jiao

2017-01-01

Full Text Available Space Information Network (SIN with backbone satellites relaying for vehicular network (VN communications is regarded as an effective strategy to provide diverse vehicular services in a seamless, efficient, and cost-effective manner in rural areas and highways. In this paper, we investigate the performance of SIN return channel cooperative communications via an amplify-and-forward (AF backbone satellite relaying for VN communications, where we assume that both of the source-destination and relay-destination links undergo Shadowed-Rician fading and the source-relay link follows Rician fading, respectively. In this SIN-assisted VN communication scenario, we first obtain the approximate statistical distributions of the equivalent end-to-end signal-to-noise ratio (SNR of the system. Then, we derive the closed-form expressions to efficiently evaluate the average symbol error rate (ASER of the system. Furthermore, the ASER expressions are taking into account the effect of satellite perturbation of the backbone relaying satellite, which reveal the accumulated error of the antenna pointing error. Finally, simulation results are provided to verify the accuracy of our theoretical analysis and show the impact of various parameters on the system performance.

Prediction of backbone dihedral angles and protein secondary structure using support vector machines

Directory of Open Access Journals (Sweden)

Hirst Jonathan D

2009-12-01

Full Text Available Abstract Background The prediction of the secondary structure of a protein is a critical step in the prediction of its tertiary structure and, potentially, its function. Moreover, the backbone dihedral angles, highly correlated with secondary structures, provide crucial information about the local three-dimensional structure. Results We predict independently both the secondary structure and the backbone dihedral angles and combine the results in a loop to enhance each prediction reciprocally. Support vector machines, a state-of-the-art supervised classification technique, achieve secondary structure predictive accuracy of 80% on a non-redundant set of 513 proteins, significantly higher than other methods on the same dataset. The dihedral angle space is divided into a number of regions using two unsupervised clustering techniques in order to predict the region in which a new residue belongs. The performance of our method is comparable to, and in some cases more accurate than, other multi-class dihedral prediction methods. Conclusions We have created an accurate predictor of backbone dihedral angles and secondary structure. Our method, called DISSPred, is available online at http://comp.chem.nottingham.ac.uk/disspred/.

Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein.

Science.gov (United States)

Sharma, Monika; Anirudh, C R

2017-10-03

STAR proteins are evolutionary conserved mRNA-binding proteins that post-transcriptionally regulate gene expression at all stages of RNA metabolism. These proteins possess conserved STAR domain that recognizes identical RNA regulatory elements as YUAAY. Recently reported crystal structures show that STAR domain is composed of N-terminal QUA1, K-homology domain (KH) and C-terminal QUA2, and mRNA binding is mediated by KH-QUA2 domain. Here, we present simulation studies done to investigate binding of mRNA to STAR protein, mammalian Quaking protein (QKI). We carried out conventional MD simulations of STAR domain in presence and absence of mRNA, and studied the impact of mRNA on the stability, dynamics and underlying allosteric mechanism of STAR domain. Our unbiased simulations results show that presence of mRNA stabilizes the overall STAR domain by reducing the structural deviations, correlating the 'within-domain' motions, and maintaining the native contacts information. Absence of mRNA not only influenced the essential modes of motion of STAR domain, but also affected the connectivity of networks within STAR domain. We further explored the dissociation of mRNA from STAR domain using umbrella sampling simulations, and the results suggest that mRNA binding to STAR domain occurs in multi-step: first conformational selection of mRNA backbone conformations, followed by induced fit mechanism as nucleobases interact with STAR domain.

Effect of PEG and mPEG-anthracene on tRNA aggregation and particle formation.

Science.gov (United States)

Froehlich, E; Mandeville, J S; Arnold, D; Kreplak, L; Tajmir-Riahi, H A

2012-01-09

Poly(ethylene glycol) (PEG) and its derivatives are synthetic polymers with major applications in gene and drug delivery systems. Synthetic polymers are also used to transport miRNA and siRNA in vitro. We studied the interaction of tRNA with several PEGs of different compositions, such as PEG 3350, PEG 6000, and mPEG-anthracene under physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the PEG binding mode, the binding constant, and the effects of polymer complexation on tRNA stability, aggregation, and particle formation. Structural analysis showed that PEG-tRNA interaction occurs via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(PEG 3350-tRNA)= 1.9 (±0.5) × 10(4) M(-1), K(PEG 6000-tRNA) = 8.9 (±1) × 10(4) M(-1), and K(mPEG-anthracene)= 1.2 (±0.40) × 10(3) M(-1) show stronger polymer-RNA complexation by PEG 6000 and by PEG 3350 than the mPEG-anthracene. AFM imaging showed that PEG complexes contain on average one tRNA with PEG 3350, five tRNA with PEG 6000, and ten tRNA molecules with mPEG-anthracene. tRNA aggregation and particle formation occurred at high polymer concentrations, whereas it remains in A-family structure.

In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

Directory of Open Access Journals (Sweden)

Ivone Un San Leong

2012-01-01

Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

Smart-Grid Backbone Network Real-Time Delay Reduction via Integer Programming.

Science.gov (United States)

Pagadrai, Sasikanth; Yilmaz, Muhittin; Valluri, Pratyush

2016-08-01

This research investigates an optimal delay-based virtual topology design using integer linear programming (ILP), which is applied to the current backbone networks such as smart-grid real-time communication systems. A network traffic matrix is applied and the corresponding virtual topology problem is solved using the ILP formulations that include a network delay-dependent objective function and lightpath routing, wavelength assignment, wavelength continuity, flow routing, and traffic loss constraints. The proposed optimization approach provides an efficient deterministic integration of intelligent sensing and decision making, and network learning features for superior smart grid operations by adaptively responding the time-varying network traffic data as well as operational constraints to maintain optimal virtual topologies. A representative optical backbone network has been utilized to demonstrate the proposed optimization framework whose simulation results indicate that superior smart-grid network performance can be achieved using commercial networks and integer programming.

An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.

Science.gov (United States)

Adams, Felix F; Heckl, Dirk; Hoffmann, Thomas; Talbot, Steven R; Kloos, Arnold; Thol, Felicitas; Heuser, Michael; Zuber, Johannes; Schambach, Axel; Schwarzer, Adrian

2017-09-01

RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system. Copyright © 2017 Elsevier Ltd. All rights reserved.

NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

Science.gov (United States)

Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

2015-01-01

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

Backbone dynamics of the EIAV-Tat protein from 15N relaxation studies

International Nuclear Information System (INIS)

Ejchart, A.; Herrmann, F.; Roesch, P.; Sticht, H.; Willbold, D.

1994-01-01

The work investigates the mobility of EIAV-Tat protein backbone by measuring the relaxation parameters of the 15 N nitrogens. High degree of the flexibility, non-typical of rigid, well structured proteins was shown

Efficient transformation and artificial miRNA gene silencing in Lemna minor.

Science.gov (United States)

Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

2015-01-01

Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Probing the role of backbone hydrogen bonds in protein-peptide interactions by amide-to-ester mutations

DEFF Research Database (Denmark)

Eildal, Jonas N N; Hultqvist, Greta; Balle, Thomas

2013-01-01

-protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as β-strands to form an extended antiparallel β-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the amino acid side chains...... of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding...... to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, ΔΔG = 1.3 to >3.8 kcal mol(-1...

Direct observation of backbone planarization via side-chain alignment in single bulky-substituted polythiophenes

Science.gov (United States)

Raithel, Dominic; Simine, Lena; Pickel, Sebastian; Schötz, Konstantin; Panzer, Fabian; Baderschneider, Sebastian; Schiefer, Daniel; Lohwasser, Ruth; Köhler, Jürgen; Thelakkat, Mukundan; Sommer, Michael; Köhler, Anna; Rossky, Peter J.; Hildner, Richard

2018-03-01

The backbone conformation of conjugated polymers affects, to a large extent, their optical and electronic properties. The usually flexible substituents provide solubility and influence the packing behavior of conjugated polymers in films or in bad solvents. However, the role of the side chains in determining and potentially controlling the backbone conformation, and thus the optical and electronic properties on the single polymer level, is currently under debate. Here, we investigate directly the impact of the side chains by studying the bulky-substituted poly(3-(2,5-dioctylphenyl)thiophene) (PDOPT) and the common poly(3-hexylthiophene) (P3HT), both with a defined molecular weight and high regioregularity, using low-temperature single-chain photoluminescence (PL) spectroscopy and quantum-classical simulations. Surprisingly, the optical transition energy of PDOPT is significantly (˜2,000 cm‑1 or 0.25 eV) red-shifted relative to P3HT despite a higher static and dynamic disorder in the former. We ascribe this red shift to a side-chain induced backbone planarization in PDOPT, supported by temperature-dependent ensemble PL spectroscopy. Our atomistic simulations reveal that the bulkier 2,5-dioctylphenyl side chains of PDOPT adopt a clear secondary helical structural motif and thus protect conjugation, i.e., enforce backbone planarity, whereas, for P3HT, this is not the case. These different degrees of planarity in both thiophenes do not result in different conjugation lengths, which we found to be similar. It is rather the stronger electronic coupling between the repeating units in the more planar PDOPT which gives rise to the observed spectral red shift as well as to a reduced calculated electron‑hole polarization.

Effect of oligonucleic acid (ONA) backbone features on assembly of ONA-star polymer conjugates: a coarse-grained molecular simulation study.

Science.gov (United States)

Condon, Joshua E; Jayaraman, Arthi

2017-10-04

Understanding the impact of incorporating new physical and chemical features in oligomeric DNA mimics, termed generally as "oligonucleic acids" (ONAs), on their structure and thermodynamics will be beneficial in designing novel materials for a variety of applications. In this work, we conduct coarse-grained molecular simulations of ONA-star polymer conjugates with varying ONA backbone flexibility, ONA backbone charge, and number of arms in the star polymer at a constant ONA strand volume fraction to elucidate the effect of these design parameters on the thermodynamics and assembly of multi-arm ONA-star polymer conjugates. We quantify the thermo-reversible behavior of the ONA-star polymer conjugates by quantifying the hybridization of the ONA strands in the system as a function of temperature (i.e. melting curve). Additionally, we characterize the assembly of the ONA-star polymer conjugates by tracking cluster formation and percolation as a function of temperature, as well as cluster size distribution at temperatures near the assembly transition region. The key results are as follows. The melting temperature (T m ) of the ONA strands decreases upon going from a neutral to a charged ONA backbone and upon increasing flexibility of the ONA backbone. Similar behavior is seen for the assembly transition temperature (T a ) with varying ONA backbone charge and flexibility. While the number of arms in the ONA-star polymer conjugate has a negligible effect on the ONA T m in these systems, as the number of ONA-star polymer arms increase, the assembly temperature T a increases and local ordering in the assembled state improves. By understanding how factors like ONA backbone charge, backbone flexibility, and ONA-star polymer conjugate architecture impact the behavior of ONA-star polymer conjugate systems, we can better inform how the selection of ONA chemistry will influence resulting ONA-star polymer assembly.

Effect of backbone structure on charge transport along isolated conjugated polymer chains

International Nuclear Information System (INIS)

Siebbeles, Laurens D.A.; Grozema, Ferdinand C.; Haas, Matthijs P. de; Warman, John M.

2005-01-01

Fast charge transport in conjugated polymers is essential for their application in opto-electronic devices. In the present paper, measurements and theoretical modeling of the mobility of excess charges along isolated chains of conjugated polymers in dilute solution are presented. Charge carriers were produced by irradiation of the polymer solution with 3-MeV electrons from a Van de Graaff accelerator. The mobilities of the charges along the polymer chains were obtained from time-resolved microwave conductivity measurements. The mobilities are strongly dependent on the chemical nature of the polymer backbone. Comparison of the experimental data with results from ab initio quantum mechanical calculations shows that the measured mobilities are strongly limited by torsional disorder, chemical defects and chain ends. Improvement of the structure of polymer backbones is therefore expected to significantly enhance the performance of these materials in 'plastic electronics'

Generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation

Directory of Open Access Journals (Sweden)

Wang Genping

2016-09-01

Full Text Available Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154 and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154 were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of ‘clean’ GM wheat containing only the foreign genes of agronomic importance.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Science.gov (United States)

Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D; Xia, Lan-Qin

2016-01-01

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium -mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Aromatic Copolyamides with Anthrazoline Units in the Backbone: Synthesis, Characterization, Pervaporation Application

Directory of Open Access Journals (Sweden)

Galina A. Polotskaya

2016-10-01

Full Text Available Copolyamides with anthrazoline units in the backbone (coPA were synthesized and dense nonporous films were prepared by solvent evaporation. Glass transition temperature, density, and fractional free volume were determined for the dense nonporous films composed of polyamide and two of its copolymers containing 20 and 30 mol % anthrazoline units in the backbone. Transport properties of the polymer films were estimated by sorption and pervaporation tests toward methanol, toluene, and their mixtures. An increase in anthrazoline fragments content leads to an increasing degree of methanol sorption but to a decreasing degree of toluene sorption. Pervaporation of a methanol–toluene mixture was studied over a wide range of feed concentration (10–90 wt % methanol. Maximal separation factor was observed for coPA-20 containing 20 mol % fragments with anthrazoline units; maximal total flux was observed for coPA-30 with the highest fractional free volume.

Altered endoribonuclease activity of apurinic/apyrimidinic endonuclease 1 variants identified in the human population.

Directory of Open Access Journals (Sweden)

Wan Cheol Kim

Full Text Available Apurinic/apyrimidinic endonuclease 1 (APE1 is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. APE1 also has 3'-5' DNA exonuclease and 3' DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76-85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3' of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60-80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

CORBA and MPI-based 'backbone' for coupling advanced simulation tools

International Nuclear Information System (INIS)

Seydaliev, M.; Caswell, D.

2014-01-01

There is a growing international interest in using coupled, multidisciplinary computer simulations for a variety of purposes, including nuclear reactor safety analysis. Reactor behaviour can be modeled using a suite of computer programs simulating phenomena or predicting parameters that can be categorized into disciplines such as Thermalhydraulics, Neutronics, Fuel, Fuel Channels, Fission Product Release and Transport, Containment and Atmospheric Dispersion, and Severe Accident Analysis. Traditionally, simulations used for safety analysis individually addressed only the behaviour within a single discipline, based upon static input data from other simulation programs. The limitation of using a suite of stand-alone simulations is that phenomenological interdependencies or temporal feedback between the parameters calculated within individual simulations cannot be adequately captured. To remove this shortcoming, multiple computer simulations for different disciplines must exchange data during runtime to address these interdependencies. This article describes the concept of a new framework, which we refer to as the 'Backbone', to provide the necessary runtime exchange of data. The Backbone, currently under development at AECL for a preliminary feasibility study, is a hybrid design using features taken from the Common Object Request Broker Architecture (CORBA), a standard defined by the Object Management Group, and the Message Passing Interface (MPI), a standard developed by a group of researchers from academia and industry. Both have well-tested and efficient implementations, including some that are freely available under the GNU public licenses. The CORBA component enables individual programs written in different languages and running on different platforms within a network to exchange data with each other, thus behaving like a single application. MPI provides the process-to-process intercommunication between these programs. This paper outlines the different CORBA and

Ca-C backbone fragmentation dominates in electron detachment dissociation of gas-phase polypeptide polyanions

DEFF Research Database (Denmark)

Kjeldsen, Frank; Silivra, Oleg A; Ivonin, Igor A

2005-01-01

the dissociation of oxidized radical anions [M-nH]((n-1)-*. We demonstrate that C(alpha)-C cleavages, which are otherwise rarely observed in tandem mass spectrometry, can account for most of the backbone fragmentation, with even-electron x fragments dominating over radical a* ions. Ab initio calculations at the B3...... LYP level of theory with the 6-311+G(2 p,2 d)//6-31+G(d,p) basis set suggested a unidirectional mechanism for EDD (cleavage always N-terminal to the radical site), with a*, x formation being favored over a, x* fragmentation by 74.2 kJ mol(-1). Thus, backbone C(alpha)-C bonds N-terminal to proline...

Effects of backbone conformation and surface texture of polyimide alignment film on the pretilt angle of liquid crystals

International Nuclear Information System (INIS)

Chang, Chi-Jung; Chou, Ray-Lin; Lin, Yu-Chi; Liang, Bau-Jy; Chen, Jyun-Ji

2011-01-01

Polyimides (PIs) with different inclination angle of polymer backbones, together with polar hydroxyl group and/or nonpolar trifluoromethyl group at various sites of the backbone were synthesized and used as liquid crystal alignment layers. The molecular conformation, surface chemistry, surface energy, surface morphology, and pretilt angle of the PI film were investigated. The distributions of fluorinated group and hydroxyl group at different depths of the PI surfaces were analyzed by X-ray photoelectron spectroscopy. Effects of the conformation of the PI molecular backbone on the surface morphology of the rubbed PI layer, the pretilt angle and surface energy of the alignment film were studied. The PI which contains both nonpolar fluorinated groups sticking out of the surface and the polar hydroxyl groups on the surface exhibits high pretilt angle.

Electric field induced localization phenomena in a ladder network with superlattice configuration: Effect of backbone environment

Energy Technology Data Exchange (ETDEWEB)

Dutta, Paramita; Karmakar, S. N. [Condensed Matter Physics Division, Saha Institute of Nuclear Physics, Sector-I, Block-AF, Bidhannagar, Kolkata-700 064 (India); Maiti, Santanu K., E-mail: santanu.maiti@isical.ac.in [Physics and Applied Mathematics Unit, Indian Statistical Institute, 203 Barrackpore Trunk Road, Kolkata-700 108 (India)

2014-09-15

Electric field induced localization properties of a tight-binding ladder network in presence of backbone sites are investigated. Based on Green's function formalism we numerically calculate two-terminal transport together with density of states for different arrangements of atomic sites in the ladder and its backbone. Our results lead to a possibility of getting multiple mobility edges which essentially plays a switching action between a completely opaque to fully or partly conducting region upon the variation of system Fermi energy, and thus, support in fabricating mesoscopic or DNA-based switching devices.

Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

Science.gov (United States)

Watters, Kyle E; Lucks, Julius B

2016-01-01

Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

Electrostatic Origin of the Red Solvatochromic Shift of DFHBDI in RNA Spinach.

Science.gov (United States)

Bose, Samik; Chakrabarty, Suman; Ghosh, Debashree

2017-05-11

Interactions with the environment tune the spectral properties of biological chromophores, e.g., fluorescent proteins. Understanding the relative contribution of the various types of noncovalent interactions in the spectral shifts can provide rational design principles toward developing new fluorescent probes. In this work, we investigate the origin of the red shift in the absorption spectra of the difluoro hydroxybenzylidene dimethyl imidazolinone (DFHBDI) chromophore in RNA spinach as compared to the aqueous solution. We systematically decompose the effects of various components of interactions, namely, stacking, hydrogen bonding, and long-range electrostatics, in order to elucidate the relative role of these interactions in the observed spectral behavior. We find that the absorption peak of DFHBDI is red-shifted by ∼0.35 eV in RNA relative to the aqueous solution. Earlier proposals from Huang and co-workers have implicated the stacking interactions between DFHBDI and nucleic acid bases to be the driving force behind the observed red shift. In contrast, our findings reveal that the long-range electrostatic interactions between DFHBDI and negatively charged RNA make the most significant contribution. Moreover, we notice that the opposing electrostatic fields due to the RNA backbone and the polarized water molecules around the RNA give rise to the resultant red shift. Our results emphasize the effect of strong heterogeneity in the various environmental factors that might be competing with each other.

Influence of Backbone Fluorination in Regioregular Poly(3-alkyl-4-fluoro)thiophenes

KAUST Repository

Fei, Zhuping

2015-06-03

© 2015 American Chemical Society. We report two strategies toward the synthesis of 3-alkyl-4-fluorothiophenes containing straight (hexyl and octyl) and branched (2-ethylhexyl) alkyl groups. We demonstrate that treatment of the dibrominated monomer with 1 equiv of alkyl Grignard reagent leads to the formation of a single regioisomer as a result of the pronounced directing effect of the fluorine group. Polymerization of the resulting species affords highly regioregular poly(3-alkyl-4-fluoro)thiophenes. Comparison of their properties to those of the analogous non-fluorinated polymers shows that backbone fluorination leads to an increase in the polymer ionization potential without a significant change in optical band gap. Fluorination also results in an enhanced tendency to aggregate in solution, which is ascribed to a more co-planar backbone on the basis of Raman and DFT calculations. Average charge carrier mobilities in field-effect transistors are found to increase by up to a factor of 5 for the fluorinated polymers.

Data Acquisition Backbone Core DABC

International Nuclear Information System (INIS)

Adamczewski, J; Essel, H G; Kurz, N; Linev, S

2008-01-01

For the new experiments at FAIR new concepts of data acquisition systems have to be developed like the distribution of self-triggered, time stamped data streams over high performance networks for event building. The Data Acquisition Backbone Core (DABC) is a software package currently under development for FAIR detector tests, readout components test, and data flow investigations. All kinds of data channels (front-end systems) are connected by program plug-ins into functional components of DABC like data input, combiner, scheduler, event builder, analysis and storage components. After detailed simulations real tests of event building over a switched network (InfiniBand clusters with up to 110 nodes) have been performed. With the DABC software more than 900 MByte/s input and output per node can be achieved meeting the most demanding requirements. The software is ready for the implementation of various test beds needed for the final design of data acquisition systems at FAIR. The development of key components is supported by the FutureDAQ project of the European Union (FP6 I3HP JRA1)

Inferential backbone assignment for sparse data

International Nuclear Information System (INIS)

Vitek, Olga; Bailey-Kellogg, Chris; Craig, Bruce; Vitek, Jan

2006-01-01

This paper develops an approach to protein backbone NMR assignment that effectively assigns large proteins while using limited sets of triple-resonance experiments. Our approach handles proteins with large fractions of missing data and many ambiguous pairs of pseudoresidues, and provides a statistical assessment of confidence in global and position-specific assignments. The approach is tested on an extensive set of experimental and synthetic data of up to 723 residues, with match tolerances of up to 0.5 ppm for C α and C β resonance types. The tests show that the approach is particularly helpful when data contain experimental noise and require large match tolerances. The keys to the approach are an empirical Bayesian probability model that rigorously accounts for uncertainty in the data at all stages in the analysis, and a hybrid stochastic tree-based search algorithm that effectively explores the large space of possible assignments

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...

Backbone dynamics of the human CC-chemokine eotaxin

Energy Technology Data Exchange (ETDEWEB)

Ye Jiqing; Mayer, Kristen L.; Stone, Martin J. [Indiana University, Department of Chemistry (United States)

1999-10-15

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. {sup 15}N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. {sup 15}N longitudinal (R{sub 1}) and transverse (R{sub 2}) auto relaxation rates, heteronuclear {sup 1}H-{sup 15}N steady-state NOEs, and transverse cross-relaxation rates ({eta}{sub xy}) were obtained at 30 deg. C for all resolved backbone secondary amide groups using {sup 1} H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time ({tau}{sub m}) is 5.09{+-}0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D{sub parallel}/D{sub perpendicular}) is 0.81{+-}0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1-19), the C-terminus (residues 68-73) and the loop connecting the first two {beta}-strands (residues 30-37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond-millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.

Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization.

Science.gov (United States)

Caffrey, Leah M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

2016-10-10

A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (siRNA), is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit, and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that fluorescein isothiocyanate (FITC)-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments will not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.

Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops

Directory of Open Access Journals (Sweden)

Gendrault-Jacquemard A

2005-07-01

Full Text Available Abstract Background Public databases now contain multitude of complete bacterial genomes, including several genomes of the same species. The available data offers new opportunities to address questions about bacterial genome evolution, a task that requires reliable fine comparison data of closely related genomes. Recent analyses have shown, using pairwise whole genome alignments, that it is possible to segment bacterial genomes into a common conserved backbone and strain-specific sequences called loops. Results Here, we generalize this approach and propose a strategy that allows systematic and non-biased genome segmentation based on multiple genome alignments. Segmentation analyses, as applied to 13 different bacterial species, confirmed the feasibility of our approach to discern the 'mosaic' organization of bacterial genomes. Segmentation results are available through a Web interface permitting functional analysis, extraction and visualization of the backbone/loops structure of documented genomes. To illustrate the potential of this approach, we performed a precise analysis of the mosaic organization of three E. coli strains and functional characterization of the loops. Conclusion The segmentation results including the backbone/loops structure of 13 bacterial species genomes are new and available for use by the scientific community at the URL: http://genome.jouy.inra.fr/mosaic.

Treatment Results of Injuries of Thoracic and Lumbar Backbone Departments at Osteoporosis Patients

Directory of Open Access Journals (Sweden)

D.Y. Sumin

2009-06-01

Full Text Available Information relates to radiologic (computer tomography manifestations providing the visualization of thoracic and lumbar backbone department injuries at osteoporotic patients. Contemporary methods of transcutaneous and trans-pedicle vertebroplasty with bone cement allows to obtain a stable positive healing effect against such pathologies.

Long-Range Vibrational Dynamics Are Directed by Watson-Crick Base Pairing in Duplex DNA.

Science.gov (United States)

Hithell, Gordon; Shaw, Daniel J; Donaldson, Paul M; Greetham, Gregory M; Towrie, Michael; Burley, Glenn A; Parker, Anthony W; Hunt, Neil T

2016-05-05

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Sulfation and cation effects on the conformational properties of the glycan backbone of chondroitin sulfate disaccharides.

Science.gov (United States)

Faller, Christina E; Guvench, Olgun

2015-05-21

Chondroitin sulfate (CS) is one of several glycosaminoglycans that are major components of proteoglycans. A linear polymer consisting of repeats of the disaccharide -4GlcAβ1-3GalNAcβ1-, CS undergoes differential sulfation resulting in five unique sulfation patterns. Because of the dimer repeat, the CS glycosidic "backbone" has two distinct sets of conformational degrees of freedom defined by pairs of dihedral angles: (ϕ1, ψ1) about the β1-3 glycosidic linkage and (ϕ2, ψ2) about the β1-4 glycosidic linkage. Differential sulfation and the possibility of cation binding, combined with the conformational flexibility and biological diversity of CS, complicate experimental efforts to understand CS three-dimensional structures at atomic resolution. Therefore, all-atom explicit-solvent molecular dynamics simulations with Adaptive Biasing Force sampling of the CS backbone were applied to obtain high-resolution, high-precision free energies of CS disaccharides as a function of all possible backbone geometries. All 10 disaccharides (β1-3 vs β1-4 linkage × five different sulfation patterns) were studied; additionally, ion effects were investigated by considering each disaccharide in the presence of either neutralizing sodium or calcium cations. GlcAβ1-3GalNAc disaccharides have a single, broad, thermodynamically important free-energy minimum, whereas GalNAcβ1-4GlcA disaccharides have two such minima. Calcium cations but not sodium cations bind to the disaccharides, and binding is primarily to the GlcA -COO(-) moiety as opposed to sulfate groups. This binding alters the glycan backbone thermodynamics in instances where a calcium cation bound to -COO(-) can act to bridge and stabilize an interaction with an adjacent sulfate group, whereas, in the absence of this cation, the proximity of a sulfate group to -COO(-) results in two like charges being both desolvated and placed adjacent to each other and is found to be destabilizing. In addition to providing information

Life estimation and analysis of dielectric strength, hydrocarbon backbone and oxidation of high voltage multi stressed EPDM composites

Science.gov (United States)

Khattak, Abraiz; Amin, Muhammad; Iqbal, Muhammad; Abbas, Naveed

2018-02-01

Micro and nanocomposites of ethylene propylene diene monomer (EPDM) are recently studied for different characteristics. Study on life estimation and effects of multiple stresses on its dielectric strength and backbone scission and oxidation is also vital for endorsement of these composites for high voltage insulation and other outdoor applications. In order to achieve these goals, unfilled EPDM and its micro and nanocomposites are prepared at 23 phr micro silica and 6 phr nanosilica loadings respectively. Prepared samples are energized at 2.5 kV AC voltage and subjected for a long time to heat, ultraviolet radiation, acid rain, humidity and salt fog in accelerated manner in laboratory. Dielectric strength, leakage current and intensity of saturated backbone and carbonyl group are periodically measured. Loss in dielectric strength, increase in leakage current and backbone degradation and oxidation were observed in all samples. These effects were least in the case of EPDM nanocomposite. The nanocomposite sample also demonstrated longest shelf life.

Analysis of electric moments of RNA-binding proteins: implications for mechanism and prediction

Directory of Open Access Journals (Sweden)

Sarai Akinori

2011-02-01

Full Text Available Abstract Background Protein-RNA interactions play important role in many biological processes such as gene regulation, replication, protein synthesis and virus assembly. Although many structures of various types of protein-RNA complexes have been determined, the mechanism of protein-RNA recognition remains elusive. We have earlier shown that the simplest electrostatic properties viz. charge, dipole and quadrupole moments, calculated from backbone atomic coordinates of proteins are biased relative to other proteins, and these quantities can be used to identify DNA-binding proteins. Closely related, RNA-binding proteins are investigated in this study. In particular, discrimination between various types of RNA-binding proteins, evolutionary conservation of these bulk electrostatic features and effect of conformational changes by complex formation are investigated. Basic binding mechanism of a putative RNA-binding protein (HI1333 from Haemophilus influenza is suggested as a potential application of this study. Results We found that similar to DNA-binding proteins (DBPs, RNA-binding proteins (RBPs also show significantly higher values of electric moments. However, higher moments in RBPs are found to strongly depend on their functional class: proteins binding to ribosomal RNA (rRNA constitute the only class with all three of the properties (charge, dipole and quadrupole moments being higher than control proteins. Neural networks were trained using leave-one-out cross-validation to predict RBPs from control data as well as pair-wise classification capacity between proteins binding to various RNA types. RBPs and control proteins reached up to 78% accuracy measured by the area under the ROC curve. Proteins binding to rRNA are found to be best distinguished (AUC = 79%. Changes in dipole and quadrupole moments between unbound and bound structures were small and these properties are found to be robust under complex formation. Conclusions Bulk electric

Protein backbone angle restraints from searching a database for chemical shift and sequence homology

Energy Technology Data Exchange (ETDEWEB)

Cornilescu, Gabriel; Delaglio, Frank; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

1999-03-15

Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C{alpha}, 13C{beta}, 13C', 1H{alpha} and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar {phi} and {psi} backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 deg. Approximately 3% of the predictions made by TALOS are found to be in error.

Controlled Conjugated Backbone Twisting for an Increased Open-Circuit Voltage while Having a High Short-Circuit Current in Poly(hexylthiophene) Derivatives

KAUST Repository

Ko, Sangwon

2012-03-21

Conjugated polymers with nearly planar backbones have been the most commonly investigated materials for organic-based electronic devices. More twisted polymer backbones have been shown to achieve larger open-circuit voltages in solar cells, though with decreased short-circuit current densities. We systematically impose twists within a family of poly(hexylthiophene)s and examine their influence on the performance of polymer:fullerene bulk heterojunction (BHJ) solar cells. A simple chemical modification concerning the number and placement of alkyl side chains along the conjugated backbone is used to control the degree of backbone twisting. Density functional theory calculations were carried out on a series of oligothiophene structures to provide insights on how the sterically induced twisting influences the geometric, electronic, and optical properties. Grazing incidence X-ray scattering measurements were performed to investigate how the thin-film packing structure was affected. The open-circuit voltage and charge-transfer state energy of the polymer:fullerene BHJ solar cells increased substantially with the degree of twist induced within the conjugated backbone-due to an increase in the polymer ionization potential-while the short-circuit current decreased as a result of a larger optical gap and lower hole mobility. A controlled, moderate degree of twist along the poly(3,4-dihexyl-2,2′:5′,2′′- terthiophene) (PDHTT) conjugated backbone led to a 19% enhancement in the open-circuit voltage (0.735 V) vs poly(3-hexylthiophene)-based devices, while similar short-circuit current densities, fill factors, and hole-carrier mobilities were maintained. These factors resulted in a power conversion efficiency of 4.2% for a PDHTT:[6,6]-phenyl-C 71-butyric acid methyl ester (PC 71BM) blend solar cell without thermal annealing. This simple approach reveals a molecular design avenue to increase open-circuit voltage while retaining the short-circuit current. © 2012 American

Controlled conjugated backbone twisting for an increased open-circuit voltage while having a high short-circuit current in poly(hexylthiophene) derivatives.

Science.gov (United States)

Ko, Sangwon; Hoke, Eric T; Pandey, Laxman; Hong, Sanghyun; Mondal, Rajib; Risko, Chad; Yi, Yuanping; Noriega, Rodrigo; McGehee, Michael D; Brédas, Jean-Luc; Salleo, Alberto; Bao, Zhenan

2012-03-21

Conjugated polymers with nearly planar backbones have been the most commonly investigated materials for organic-based electronic devices. More twisted polymer backbones have been shown to achieve larger open-circuit voltages in solar cells, though with decreased short-circuit current densities. We systematically impose twists within a family of poly(hexylthiophene)s and examine their influence on the performance of polymer:fullerene bulk heterojunction (BHJ) solar cells. A simple chemical modification concerning the number and placement of alkyl side chains along the conjugated backbone is used to control the degree of backbone twisting. Density functional theory calculations were carried out on a series of oligothiophene structures to provide insights on how the sterically induced twisting influences the geometric, electronic, and optical properties. Grazing incidence X-ray scattering measurements were performed to investigate how the thin-film packing structure was affected. The open-circuit voltage and charge-transfer state energy of the polymer:fullerene BHJ solar cells increased substantially with the degree of twist induced within the conjugated backbone--due to an increase in the polymer ionization potential--while the short-circuit current decreased as a result of a larger optical gap and lower hole mobility. A controlled, moderate degree of twist along the poly(3,4-dihexyl-2,2':5',2''-terthiophene) (PDHTT) conjugated backbone led to a 19% enhancement in the open-circuit voltage (0.735 V) vs poly(3-hexylthiophene)-based devices, while similar short-circuit current densities, fill factors, and hole-carrier mobilities were maintained. These factors resulted in a power conversion efficiency of 4.2% for a PDHTT:[6,6]-phenyl-C(71)-butyric acid methyl ester (PC(71)BM) blend solar cell without thermal annealing. This simple approach reveals a molecular design avenue to increase open-circuit voltage while retaining the short-circuit current.

First-line HIV treatment: evaluation of backbone choice and its budget impact

Directory of Open Access Journals (Sweden)

Orietta Zaniolo

2013-06-01

Full Text Available OBJECTIVE: The gradual increase of persons living with HIV, mainly due to the reduced mortality achieved with effective antiretroviral therapies, calls for increased rationality and awareness in health resources consumption also during the early illness phases. Aim of this work is the estimation of the budget impact related to the variation in backbone prescribing trends in naïve patients.METHODS: Target population is the number of patients starting antiretroviral therapy each year, according to the Italian HIV surveillance registry, excluding patients receiving non-authorized or non-recommended regimens. We modeled 3-year mortality and durability rates on a dynamic cohort, basing on international literature. A prevalent patients analysis has also been conducted, for which the model is fed by a closed cohort consisting of all the patients without experience of virologic failure. The aim of this collateral analysis is to estimate the difference in current annual expenditures if the past prescription trends for patients starting therapy would have led to the evaluated hypothetical scenarios. Current Italian market shares of triple regimens containing first-choice or alternative backbones (tenofovir/emtricitabine, abacavir/lamivudine, tenofovir/lamivudine and zidovudine/lamivudine are compared to three hypothetical scenarios (base-case, minimum and maximum in which increasing shares of patients eligible to abacavir/lamivudine start first line treatment with this backbone. Annual cost for each regimen comprises drugs acquisition under hospital pricing rules, monitoring exams and preventive tests, valued basing on regional reimbursement tariffs.RESULTS: According to current prescribing trends, in the next three years about 13,000 patients starting HIV therapy will receive tenofovir/emtricitabine (83% of the target population, and minor portions other regimens (9% abacavir/lamivudine, 8% zidovudine/lamivudine. Patients that would be eligible to

Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

International Nuclear Information System (INIS)

Headlam, H.A.; Davies, M.J.; Mortimer, A.; Easton, C.J.

2000-01-01

Full text: Exposure of proteins to radicals in the presence of O 2 brings about multiple changes including side-chain oxidation, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (e.g. hydroperoxides and 3,4-dihydroxyphenylalanine). All of these processes can result in loss of structural or enzymatic activity. The mechanisms that give rise to backbone cleavage are only partly understood. Whilst it is known that direct hydrogen atom abstraction at a-carbon sites gives backbone cleavages it has also been proposed that initial attack at side-chain sites might also give rise to backbone cleavage. In this study we have examined whether initial attack at the β- (C-3) position can give rise to α-carbon radicals (and hence backbone cleavage) via the formation, and subsequent β- scission, of C-3 alkoxyl radicals. This process has been observed previously with protected amino acids in organic solvents (J. Chem. Soc. Perkin Trans. 2, 1997, 503-507) but the occurrence of such reactions with proteins in aqueous solution has not been explored. Alkoxyl radicals were generated at the C-3 position of a variety of protected amino acids and small peptides by two methods: metal-ion catalysed decomposition of hydroperoxides formed as a result of γ-radiolysis in the presence of O 2 , and UV photolysis of nitrate esters. In most cases radicals have been detected by EPR spectroscopy using nitroso and nitrone spin traps, which can be assigned by comparison with literature data to α-carbon radicals; in some case assignments were confirmed by the generation of the putative species by other routes. With Ala peptide hydroperoxides and nitrate esters, and MNP as the spin trap, the major radical detected in each case has been assigned to the adduct of an α-carbon radical with partial structure - NH- . CH-C(O) - consistent with the rapid occurrence of the above

Mars - robust automatic backbone assignment of proteins

International Nuclear Information System (INIS)

Jung, Young-Sang; Zweckstetter, Markus

2004-01-01

MARS a program for robust automatic backbone assignment of 13 C/ 15 N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only 13 C α / 13 C β connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment

Improving VANETs Connectivity with a Totally Ad Hoc Living Mobile Backbone

Directory of Open Access Journals (Sweden)

Joilson Alves Junior

2015-01-01

Full Text Available The vehicular ad hoc network (VANET for intelligent transportation systems is an emerging concept to improve transportation security, reliability, and management. The network behavior can be totally different in topological aspects because of the mobility of vehicular nodes. The topology can be fully connected when the flow of vehicles is high and may have low connectivity or be invalid when the flow of vehicles is low or unbalanced. In big cities, the metropolitan buses that travel on exclusive lanes may be used to set up a metropolitan vehicular data network (backbone, raising the connectivity among the vehicles. Therefore, this paper proposes the implementation of a living mobile backbone, totally ad hoc (MOB-NET, which will provide infrastructure and raise the network connectivity. In order to show the viability of MOB-NET, statistical analyses were made with real data of express buses that travel through exclusive lanes, besides evaluations through simulations and analytic models. The statistic, analytic, and simulation results prove that the buses that travel through exclusive lanes can be used to build a communication network totally ad hoc and provide connectivity in more than 99% of the time, besides raising the delivery rate up to 95%.

Comparing the Reliability of Regular Topologies on a Backbone Network. A Case Study

DEFF Research Database (Denmark)

Cecilio, Sergio Labeage; Gutierrez Lopez, Jose Manuel; Riaz, M. Tahir

2009-01-01

The aim of this paper is to compare the reliability of regular topologies on a backbone network. The study is focused on a large-scale fiberoptic network. Different regular topological solutions as single ring, double ring or 4-Regular grid are applied to the case study, and compared in terms...

The extension of a DNA double helix by an additional Watson-Crick base pair on the same backbone

DEFF Research Database (Denmark)

Kumar, P.; Sharma, P. K.; Madsen, Charlotte S.

2013-01-01

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand.......Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand....

Design of an IPTV Multicast System for Internet Backbone Networks

Directory of Open Access Journals (Sweden)

T. H. Szymanski

2010-01-01

Full Text Available The design of an IPTV multicast system for the Internet backbone network is presented and explored through extensive simulations. In the proposed system, a resource reservation algorithm such as RSVP, IntServ, or DiffServ is used to reserve resources (i.e., bandwidth and buffer space in each router in an IP multicast tree. Each router uses an Input-Queued, Output-Queued, or Crosspoint-Queued switch architecture with unity speedup. A recently proposed Recursive Fair Stochastic Matrix Decomposition algorithm used to compute near-perfect transmission schedules for each IP router. The IPTV traffic is shaped at the sources using Application-Specific Token Bucker Traffic Shapers, to limit the burstiness of incoming network traffic. The IPTV traffic is shaped at the destinations using Application-Specific Playback Queues, to remove residual network jitter and reconstruct the original bursty IPTV video streams at each destination. All IPTV traffic flows are regenerated at the destinations with essentially zero delay jitter and essentially-perfect QoS. The destination nodes deliver the IPTV streams to the ultimate end users using the same IPTV multicast system over a regional Metropolitan Area Network. It is shown that all IPTV traffic is delivered with essentially-perfect end-to-end QoS, with deterministic bounds on the maximum delay and jitter on each video frame. Detailed simulations of an IPTV distribution system, multicasting several hundred high-definition IPTV video streams over several essentially saturated IP backbone networks are presented.

The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain.

Directory of Open Access Journals (Sweden)

Søren W. Pedersen

Full Text Available Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

Tritium containing polymers having a polymer backbone substantially void of tritium

Science.gov (United States)

Jensen, G.A.; Nelson, D.A.; Molton, P.M.

1992-03-31

A radioluminescent light source comprises a solid mixture of a phosphorescent substance and a tritiated polymer. The solid mixture forms a solid mass having length, width, and thickness dimensions, and is capable of self-support. In one aspect of the invention, the phosphorescent substance comprises solid phosphor particles supported or surrounded within a solid matrix by a tritium containing polymer. The tritium containing polymer comprises a polymer backbone which is essentially void of tritium. 2 figs.

Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks

Energy Technology Data Exchange (ETDEWEB)

Shen Yang; Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

2013-07-15

A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, {>=}90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed ({phi}, {psi}) torsion angles of ca 12 Masculine-Ordinal-Indicator . TALOS-N also reports sidechain {chi}{sup 1} rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.

Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex

Directory of Open Access Journals (Sweden)

Nancy F. Ramia

2014-12-01

Full Text Available Summary: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. : Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

The determinants of bond angle variability in protein/peptide backbones: A comprehensive statistical/quantum mechanics analysis.

Science.gov (United States)

Improta, Roberto; Vitagliano, Luigi; Esposito, Luciana

2015-11-01

The elucidation of the mutual influence between peptide bond geometry and local conformation has important implications for protein structure refinement, validation, and prediction. To gain insights into the structural determinants and the energetic contributions associated with protein/peptide backbone plasticity, we here report an extensive analysis of the variability of the peptide bond angles by combining statistical analyses of protein structures and quantum mechanics calculations on small model peptide systems. Our analyses demonstrate that all the backbone bond angles strongly depend on the peptide conformation and unveil the existence of regular trends as function of ψ and/or φ. The excellent agreement of the quantum mechanics calculations with the statistical surveys of protein structures validates the computational scheme here employed and demonstrates that the valence geometry of protein/peptide backbone is primarily dictated by local interactions. Notably, for the first time we show that the position of the H(α) hydrogen atom, which is an important parameter in NMR structural studies, is also dependent on the local conformation. Most of the trends observed may be satisfactorily explained by invoking steric repulsive interactions; in some specific cases the valence bond variability is also influenced by hydrogen-bond like interactions. Moreover, we can provide a reliable estimate of the energies involved in the interplay between geometry and conformations. © 2015 Wiley Periodicals, Inc.

Extracting the information backbone in online system.

Directory of Open Access Journals (Sweden)

Qian-Ming Zhang

Full Text Available Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such "less can be more" feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency.

Extracting the information backbone in online system.

Science.gov (United States)

Zhang, Qian-Ming; Zeng, An; Shang, Ming-Sheng

2013-01-01

Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity) of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such "less can be more" feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency.

Extracting the Information Backbone in Online System

Science.gov (United States)

Zhang, Qian-Ming; Zeng, An; Shang, Ming-Sheng

2013-01-01

Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity) of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such “less can be more” feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency. PMID:23690946

Solution structure of a DNA mimicking motif of an RNA aptamer against transcription factor AML1 Runt domain.

Science.gov (United States)

Nomura, Yusuke; Tanaka, Yoichiro; Fukunaga, Jun-ichi; Fujiwara, Kazuya; Chiba, Manabu; Iibuchi, Hiroaki; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Kozu, Tomoko; Sakamoto, Taiichi

2013-12-01

AML1/RUNX1 is an essential transcription factor involved in the differentiation of hematopoietic cells. AML1 binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. In a previous study, we obtained RNA aptamers against the AML1 Runt domain by systematic evolution of ligands by exponential enrichment and revealed that RNA aptamers exhibit higher affinity for the Runt domain than that for RDE and possess the 5'-GCGMGNN-3' and 5'-N'N'CCAC-3' conserved motif (M: A or C; N and N' form Watson-Crick base pairs) that is important for Runt domain binding. In this study, to understand the structural basis of recognition of the Runt domain by the aptamer motif, the solution structure of a 22-mer RNA was determined using nuclear magnetic resonance. The motif contains the AH(+)-C mismatch and base triple and adopts an unusual backbone structure. Structural analysis of the aptamer motif indicated that the aptamer binds to the Runt domain by mimicking the RDE sequence and structure. Our data should enhance the understanding of the structural basis of DNA mimicry by RNA molecules.

Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis

Czech Academy of Sciences Publication Activity Database

Bumba, Ladislav; Sviridova, E.; Kutá-Smatanová, Ivana; Řezáčová, Pavlína; Veverka, Václav

2014-01-01

Roč. 8, č. 1 (2014), s. 53-55 ISSN 1874-2718 R&D Projects: GA ČR(CZ) GAP207/11/0717; GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:61388971 ; RVO:67179843 Keywords : Neisseria meningitidis * FrpC * FrpD * backbone assignments * NMR * iron-regulated protein Subject RIV: CE - Biochemistry Impact factor: 0.760, year: 2014

Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

Science.gov (United States)

Wang, Li Kai; Zhu, Hui; Shuman, Stewart

2009-03-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3).

Routing protocol for wireless quantum multi-hop mesh backbone network based on partially entangled GHZ state

Science.gov (United States)

Xiong, Pei-Ying; Yu, Xu-Tao; Zhang, Zai-Chen; Zhan, Hai-Tao; Hua, Jing-Yu

2017-08-01

Quantum multi-hop teleportation is important in the field of quantum communication. In this study, we propose a quantum multi-hop communication model and a quantum routing protocol with multihop teleportation for wireless mesh backbone networks. Based on an analysis of quantum multi-hop protocols, a partially entangled Greenberger-Horne-Zeilinger (GHZ) state is selected as the quantum channel for the proposed protocol. Both quantum and classical wireless channels exist between two neighboring nodes along the route. With the proposed routing protocol, quantum information can be transmitted hop by hop from the source node to the destination node. Based on multi-hop teleportation based on the partially entangled GHZ state, a quantum route established with the minimum number of hops. The difference between our routing protocol and the classical one is that in the former, the processes used to find a quantum route and establish quantum channel entanglement occur simultaneously. The Bell state measurement results of each hop are piggybacked to quantum route finding information. This method reduces the total number of packets and the magnitude of air interface delay. The deduction of the establishment of a quantum channel between source and destination is also presented here. The final success probability of quantum multi-hop teleportation in wireless mesh backbone networks was simulated and analyzed. Our research shows that quantum multi-hop teleportation in wireless mesh backbone networks through a partially entangled GHZ state is feasible.

Influence of structures of polymer backbones on cooperative photoreorientation behavior of p-cyanoazobenzene side chains

DEFF Research Database (Denmark)

Han, Mina; Kidowaki, Masatoshi; Ichimura, Kunihiro

2001-01-01

Photoinduced orientational behavior of a polymethacrylate (CN6) and a polyester (p6a12) with p-cyanoazobenzene side chains was studied to reveal the structural effect of the liquid crystalline polymer backbones. Irradiation with linearly polarized W light resulted in the reorientation of the azob...

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

Science.gov (United States)

Krishnan, Rajagopal; Oh, Dennis H.

2010-01-01

Triplex-forming oligonucleotides (TFOs) with both DNA and 2′-O-methyl RNA backbones can direct psoralen photoadducts to specific DNA sequences. However, the functional consequences of these differing structures on psoralen photoreactivity are unknown. We designed TFO sequences with DNA and 2′-O-methyl RNA backbones conjugated to psoralen by 2-carbon linkers and examined their ability to bind and target damage to model DNA duplexes corresponding to sequences within the human HPRT gene. While TFO binding affinity was not dramatically affected by the type of backbone, psoralen photoreactivity was completely abrogated by the 2′-O-methyl RNA backbone. Photoreactivity was restored when the psoralen was conjugated to the RNA TFO via a 6-carbon linker. In contrast to the B-form DNA of triplexes formed by DNA TFOs, the CD spectra of triplexes formed with 2′-O-methyl RNA TFOs exhibited features of A-form DNA. These results indicate that 2′-O-methyl RNA TFOs induce a partial B-to-A transition in their target DNA sequences which may impair the photoreactivity of a conjugated psoralen and suggest that optimal design of TFOs to target DNA damage may require a balance between binding ability and drug reactivity. PMID:20725628

A Dynamic Combinatorial Approach for Identifying Side Groups that Stabilize DNA-Templated Supramolecular Self-Assemblies

Directory of Open Access Journals (Sweden)

Delphine Paolantoni

2015-02-01

Full Text Available DNA-templated self-assembly is an emerging strategy for generating functional supramolecular systems, which requires the identification of potent multi-point binding ligands. In this line, we recently showed that bis-functionalized guanidinium compounds can interact with ssDNA and generate a supramolecular complex through the recognition of the phosphodiester backbone of DNA. In order to probe the importance of secondary interactions and to identify side groups that stabilize these DNA-templated self-assemblies, we report herein the implementation of a dynamic combinatorial approach. We used an in situ fragment assembly process based on reductive amination and tested various side groups, including amino acids. The results reveal that aromatic and cationic side groups participate in secondary supramolecular interactions that stabilize the complexes formed with ssDNA.

Backbone-hydrazone-containing biodegradable copolymeric micelles for anticancer drug delivery

Energy Technology Data Exchange (ETDEWEB)

Xu, Jing; Luan, Shujuan; Qin, Benkai; Wang, Yingying; Wang, Kai; Qi, Peilan; Song, Shiyong, E-mail: pharmsong@henu.edu.cn [Henan University, Institute of Pharmacy (China)

2016-11-15

Well-defined biodegradable, pH-sensitive amphiphilic block polymers, poly(ethylene glycol)-Hyd-poly(lactic acid) (mPEG-Hyd-PLA) which have acid-cleavable linkages in their backbones, were synthesized via ring-opening polymerization initiated from hydrazone-containing macroinitiators. Introducing a hydrazone bond onto the backbone of an amphiphilic copolymer will find a broad-spectrum encapsulation of hydrophobic drugs. Dynamic light scattering (DLS) and transmission electron microscopy showed that the diblock copolymers self-assembled into stable micelles with average diameters of 100 nm. The mean diameters and size distribution of the hydrazone-containing micelles changed obviously in mildly acidic pH (multiple peaks from 1 to 202 nm appeared under a pH 4.0 condition) than in neutral, while there were no changes in the case of non-sensitive ones. Doxorubicin (DOX) and paclitaxel (PTX) were loaded with drug loading content ranging from 2.4 to 3.5 %, respectively. Interestingly, the anticancer drugs released from mPEG-Hyd-PLA micelles could also be promoted by the increased acidity. An in vitro cytotoxicity study showed that the DOX-loaded mPEG-Hyd-PLA micelles have significantly enhanced cytotoxicity against HepG2 cells compared with the non-sensitive poly(ethylene glycol)-block-poly(lactic acid) (mPEG-PLA) micelles. Confocal microscopy observation indicated that more DOX were delivered into the nuclei of cells following 6 or 12 h incubation with DOX-loaded mPEG-Hyd-PLA micelles. In vivo studies on H22-bearing Swiss mice demonstrated the superior anticancer activity of DOX-loaded mPEG-Hyd-PLA micelles over free DOX and DOX-loaded mPEG-PLA micelles. These hydrazone-containing pH-responsive degradable micelles provide a useful strategy for antitumor drug delivery.

Backbone-hydrazone-containing biodegradable copolymeric micelles for anticancer drug delivery

International Nuclear Information System (INIS)

Xu, Jing; Luan, Shujuan; Qin, Benkai; Wang, Yingying; Wang, Kai; Qi, Peilan; Song, Shiyong

2016-01-01

Well-defined biodegradable, pH-sensitive amphiphilic block polymers, poly(ethylene glycol)-Hyd-poly(lactic acid) (mPEG-Hyd-PLA) which have acid-cleavable linkages in their backbones, were synthesized via ring-opening polymerization initiated from hydrazone-containing macroinitiators. Introducing a hydrazone bond onto the backbone of an amphiphilic copolymer will find a broad-spectrum encapsulation of hydrophobic drugs. Dynamic light scattering (DLS) and transmission electron microscopy showed that the diblock copolymers self-assembled into stable micelles with average diameters of 100 nm. The mean diameters and size distribution of the hydrazone-containing micelles changed obviously in mildly acidic pH (multiple peaks from 1 to 202 nm appeared under a pH 4.0 condition) than in neutral, while there were no changes in the case of non-sensitive ones. Doxorubicin (DOX) and paclitaxel (PTX) were loaded with drug loading content ranging from 2.4 to 3.5 %, respectively. Interestingly, the anticancer drugs released from mPEG-Hyd-PLA micelles could also be promoted by the increased acidity. An in vitro cytotoxicity study showed that the DOX-loaded mPEG-Hyd-PLA micelles have significantly enhanced cytotoxicity against HepG2 cells compared with the non-sensitive poly(ethylene glycol)-block-poly(lactic acid) (mPEG-PLA) micelles. Confocal microscopy observation indicated that more DOX were delivered into the nuclei of cells following 6 or 12 h incubation with DOX-loaded mPEG-Hyd-PLA micelles. In vivo studies on H22-bearing Swiss mice demonstrated the superior anticancer activity of DOX-loaded mPEG-Hyd-PLA micelles over free DOX and DOX-loaded mPEG-PLA micelles. These hydrazone-containing pH-responsive degradable micelles provide a useful strategy for antitumor drug delivery.

Electrochemical synthesis of polyaniline in the presence of poly(amidosulfonic acid)s with different rigidity of polymer backbone and characterization of the films obtained

International Nuclear Information System (INIS)

Nekrasov, A.A.; Gribkova, O.L.; Eremina, T.V.; Isakova, A.A.; Ivanov, V.F.; Tverskoj, V.A.; Vannikov, A.V.

2008-01-01

We have studied electrochemical matrix polymerization of aniline in the presence of poly(amidosulfonic acid)s of different nature: poly(2-acrylamido-2-methyl-1-propanosulfonic acid) (PAMPSA, flexible backbone); poly(p,p'-(2,2'-disulfoacid)-diphenylene-iso-phthalamid) (i-PASA, semi-rigid backbone); poly(p,p'-(2,2'-disulfoacid)-diphelylene-tere-phthalamid) (t-PASA, rigid backbone). Also, we have investigated spectral and electrochemical properties of the films obtained, as well as their surface morphology. The matrix polymerization results in the formation of interpolymer complexes of polyaniline (PANI) and the above-cited polyacids. The acceleration of aniline electropolymerization in the presence of poly(amidosulfonic acid)s was observed due to association of aniline molecules to sulfonic groups of the polyacid and higher local concentration of protons near the polyacid backbone. The rigid-chain polyacids interfere with the normal course of the electropolymerization, which manifests itself in the changes of the shape of time dependences of absorbance and charge. Cyclic voltammetry and spectroelectrochemical experiments showed that the formation of interpolymer complex with rigid-chain polyacids distorts spectroelectrochemical characteristics of PANI. This evidently results from steric hindrances in the formation of quinoid units

Charge transport properties of poly(dA)-poly(dT) DNA in variation of backbone disorder and amplitude of base-pair twisting motion

Energy Technology Data Exchange (ETDEWEB)

Rahmi, Kinanti Aldilla, E-mail: kinanti.aldilla@ui.ac.id; Yudiarsah, Efta [Physics Department, FMIPA, Universitas Indonesia, Kampus UI Depok (Indonesia)

2016-04-19

By using tight binding Hamiltonian model, charge transport properties of poly(dA)-poly(dT) DNA in variation of backbone disorder and amplitude of base-pair twisting motion is studied. The DNA chain used is 32 base pairs long poly(dA)-poly(dT) molecule. The molecule is contacted to electrode at both ends. The influence of environment on charge transport in DNA is modeled as variation of backbone disorder. The twisting motion amplitude is taking into account by assuming that the twisting angle distributes following Gaussian distribution function with zero average and standard deviation proportional to square root of temperature and inversely proportional to the twisting motion frequency. The base-pair twisting motion influences both the onsite energy of the bases and electron hopping constant between bases. The charge transport properties are studied by calculating current using Landauer-Buttiker formula from transmission probabilities which is calculated by transfer matrix methods. The result shows that as the backbone disorder increases, the maximum current decreases. By decreasing the twisting motion frequency, the current increases rapidly at low voltage, but the current increases slower at higher voltage. The threshold voltage can increase or decrease with increasing backbone disorder and increasing twisting frequency.

Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

Science.gov (United States)

Garmann, Rees F; Comas-Garcia, Mauricio; Koay, Melissa S T; Cornelissen, Jeroen J L M; Knobler, Charles M; Gelbart, William M

2014-09-01

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of

Molecular couplings and energy exchange between DNA and water mapped by femtosecond infrared spectroscopy of backbone vibrations

Directory of Open Access Journals (Sweden)

Yingliang Liu

2017-07-01

Full Text Available Molecular couplings between DNA and water together with the accompanying processes of energy exchange are mapped via the ultrafast response of DNA backbone vibrations after OH stretch excitation of the water shell. Native salmon testes DNA is studied in femtosecond pump-probe experiments under conditions of full hydration and at a reduced hydration level with two water layers around the double helix. Independent of their local hydration patterns, all backbone vibrations in the frequency range from 940 to 1120 cm–1 display a quasi-instantaneous reshaping of the spectral envelopes of their fundamental absorption bands upon excitation of the water shell. The subsequent reshaping kinetics encompass a one-picosecond component, reflecting the formation of a hot ground state of the water shell, and a slower contribution on a time scale of tens of picoseconds. Such results are benchmarked by measurements with resonant excitation of the backbone modes, resulting in distinctly different absorption changes. We assign the fast changes of DNA absorption after OH stretch excitation to structural changes in the water shell which couple to DNA through the local electric fields. The second slower process is attributed to a flow of excess energy from the water shell into DNA, establishing a common heated ground state in the molecular ensemble. This interpretation is supported by theoretical calculations of the electric fields exerted by the water shell at different temperatures.

Protonation–deprotonation of the glycine backbone as followed by Raman scattering and multiconformational analysis

Energy Technology Data Exchange (ETDEWEB)

Hernández, Belén; Pflüger, Fernando [Groupe de Biophysique Moléculaire, UFR Santé-Médecine-Biologie Humaine, Université Paris 13, Sorbonne Paris Cité, 74 rue Marcel Cachin, 93017 Bobigny cedex (France); Kruglik, Sergei G. [Laboratoire Jean Perrin, FRE 3231, Université Pierre et Marie Curie (Paris 6), Case courrier 138, 75252 Paris Cedex 05 (France); Ghomi, Mahmoud, E-mail: mahmoud.ghomi@univ-paris13.fr [Groupe de Biophysique Moléculaire, UFR Santé-Médecine-Biologie Humaine, Université Paris 13, Sorbonne Paris Cité, 74 rue Marcel Cachin, 93017 Bobigny cedex (France)

2013-11-08

Highlights: • New pH-dependent Raman spectra in the middle wavenumber region (1800-300 cm{sup −1}). • New quantum mechanical calculations for exploring the Gly conformational landscape. • Construction of muticonformation based theoretical Raman spectra. - Abstract: Because of the absence of the side chain in its chemical structure and its well defined Raman spectra, glycine was selected here to follow its backbone protonation–deprotonation. The scan of the recorded spectra in the 1800–300 cm{sup −1} region led us to assign those obtained at pH 1, 6 and 12 to the cationic, zwitterionic and anionic species, respectively. These data complete well those previously published by Bykov et al. (2008) [16] devoted to the high wavenumber Raman spectra (>2500 cm{sup −1}). To reinforce our discussion, DFT calculations were carried out on the clusters of glycine + 5H{sub 2}O, mimicking reasonably the first hydration shell of the amino acid. Geometry optimization of 141 initial clusters, reflecting plausible combinations of the backbone torsion angles, allowed exploration of the conformational features, as well as construction of the theoretical Raman spectra by considering the most stable clusters containing each glycine species.

Adsorption of molecular brushes with polyelectrolyte backbones onto oppositely charged surfaces: A self-consistent field theory

NARCIS (Netherlands)

Feuz, L.; Leermakers, F.A.M.; Textor, M.; Borisov, O.V.

2008-01-01

The two-gradient version of the Scheutjens¿Fleer self-consistent field (SF-SCF) theory is employed to model the interaction between a molecular bottle brush with a polyelectrolyte backbone and neutral hydrophilic side chains and an oppositely charged surface. Our system mimics graft-copolymers with

Multi-source micro-friction identification for a class of cable-driven robots with passive backbone

Science.gov (United States)

Tjahjowidodo, Tegoeh; Zhu, Ke; Dailey, Wayne; Burdet, Etienne; Campolo, Domenico

2016-12-01

This paper analyses the dynamics of cable-driven robots with a passive backbone and develops techniques for their dynamic identification, which are tested on the H-Man, a planar cabled differential transmission robot for haptic interaction. The mechanism is optimized for human-robot interaction by accounting for the cost-benefit-ratio of the system, specifically by eliminating the necessity of an external force sensor to reduce the overall cost. As a consequence, this requires an effective dynamic model for accurate force feedback applications which include friction behavior in the system. We first consider the significance of friction in both the actuator and backbone spaces. Subsequently, we study the required complexity of the stiction model for the application. Different models representing different levels of complexity are investigated, ranging from the conventional approach of Coulomb to an advanced model which includes hysteresis. The results demonstrate each model's ability to capture the dynamic behavior of the system. In general, it is concluded that there is a trade-off between model accuracy and the model cost.

A fusion networking model for smart grid power distribution backbone communication network based on PTN

Directory of Open Access Journals (Sweden)

Wang Hao

2016-01-01

Full Text Available In current communication network for distribution in Chinese power grid systems, the fiber communication backbone network for distribution and TD-LTE power private wireless backhaul network of power grid are both bearing by the SDH optical transmission network, which also carries the communication network of transformer substation and main electric. As the data traffic of the distribution communication and TD-LTE power private wireless network grow rapidly in recent years, it will have a big impact with the SDH network’s bearing capacity which is mainly used for main electric communication in high security level. This paper presents a fusion networking model which use a multiple-layer PTN network as the unified bearing of the TD-LTE power private wireless backhaul network and fiber communication backbone network for distribution. Network dataflow analysis shows that this model can greatly reduce the capacity pressure of the traditional SDH network as well as ensure the reliability of the transmission of the communication network for distribution and TD-LTE power private wireless network.

Conformation-specific spectroscopy of capped glutamine-containing peptides: role of a single glutamine residue on peptide backbone preferences.

Science.gov (United States)

Walsh, Patrick S; Dean, Jacob C; McBurney, Carl; Kang, Hyuk; Gellman, Samuel H; Zwier, Timothy S

2016-04-28

The conformational preferences of a series of short, aromatic-capped, glutamine-containing peptides have been studied under jet-cooled conditions in the gas phase. This work seeks a bottom-up understanding of the role played by glutamine residues in directing peptide structures that lead to neurodegenerative diseases. Resonant ion-dip infrared (RIDIR) spectroscopy is used to record single-conformation infrared spectra in the NH stretch, amide I and amide II regions. Comparison of the experimental spectra with the predictions of calculations carried out at the DFT M05-2X/6-31+G(d) level of theory lead to firm assignments for the H-bonding architectures of a total of eight conformers of four molecules, including three in Z-Gln-OH, one in Z-Gln-NHMe, three in Ac-Gln-NHBn, and one in Ac-Ala-Gln-NHBn. The Gln side chain engages actively in forming H-bonds with nearest-neighbor amide groups, forming C8 H-bonds to the C-terminal side, C9 H-bonds to the N-terminal side, and an amide-stacked geometry, all with an extended (C5) peptide backbone about the Gln residue. The Gln side chain also stabilizes an inverse γ-turn in the peptide backbone by forming a pair of H-bonds that bridge the γ-turn and stabilize it. Finally, the entire conformer population of Ac-Ala-Gln-NHBn is funneled into a single structure that incorporates the peptide backbone in a type I β-turn, stabilized by the Gln side chain forming a C7 H-bond to the central amide group in the β-turn not otherwise involved in a hydrogen bond. This β-turn backbone structure is nearly identical to that observed in a series of X-(AQ)-Y β-turns in the protein data bank, demonstrating that the gas-phase structure is robust to perturbations imposed by the crystalline protein environment.

Polystyrene Backbone Polymers Consisting of Alkyl-Substituted Triazine Side Groups for Phosphorescent OLEDs

OpenAIRE

Salert, Beatrice Ch. D.; Wedel, Armin; Grubert, Lutz; Eberle, Thomas; Anémian, Rémi; Krueger, Hartmut

2012-01-01

This paper describes the synthesis of new electron-transporting styrene monomers and their corresponding polystyrenes all with a 2,4,6-triphenyl-1,3,5-triazine basic structure in the side group. The monomers differ in the alkyl substitution and in the meta-/paralinkage of the triazine to the polymer backbone. The thermal and spectroscopic properties of the new electron-transporting polymers are discussed in regard to their chemical structures. Phosphorescent OLEDs were prepared using the obta...

Ultra-sensitive EUV resists based on acid-catalyzed polymer backbone breaking

Science.gov (United States)

Manouras, Theodoros; Kazazis, Dimitrios; Koufakis, Eleftherios; Ekinci, Yasin; Vamvakaki, Maria; Argitis, Panagiotis

2018-03-01

The main target of the current work was to develop new sensitive polymeric materials for lithographic applications, focusing in particular to EUV lithography, the main chain of which is cleaved under the influence of photogenerated acid. Resist materials based on the cleavage of polymer main chain are in principle capable to create very small structures, to the dimensions of the monomers that they consist of. Nevertheless, in the case of the commonly used nonchemically amplified materials of this type issues like sensitivity and poor etch resistance limit their areas of application, whereas inadequate etch resistance and non- satisfactory process reliability are the usual problems encountered in acid catalysed materials based on main chain scission. In our material design the acid catalyzed chain cleavable polymers contain very sensitive moieties in their backbone while they remain intact in alkaline ambient. These newly synthesized polymers bear in addition suitable functional groups for the achievement of desirable lithographic characteristics (thermal stability, acceptable glass transition temperature, etch resistance, proper dissolution behavior, adhesion to the substrate). Our approach for achieving acceptable etch resistance, a main drawback in other main chain cleavable resists, is based on the introduction of polyaromatic hydrocarbons in the polymeric backbone, whereas the incorporation of an inorganic component further enhances the etch resistance. Single component systems can also be designed following the proposed approach by the incorporation of suitable PAGs and base quencher molecules in the main chain. Resist formulations based on a random copolymer designed according to the described rules evaluated in EUV exhibit ultrahigh sensitivity, capability for high resolution patterning and overall processing characteristics that make them strong candidates for industrial use upon further optimization.

A framework to find the logic backbone of a biological network.

Science.gov (United States)

Maheshwari, Parul; Albert, Réka

2017-12-06

Cellular behaviors are governed by interaction networks among biomolecules, for example gene regulatory and signal transduction networks. An often used dynamic modeling framework for these networks, Boolean modeling, can obtain their attractors (which correspond to cell types and behaviors) and their trajectories from an initial state (e.g. a resting state) to the attractors, for example in response to an external signal. The existing methods however do not elucidate the causal relationships between distant nodes in the network. In this work, we propose a simple logic framework, based on categorizing causal relationships as sufficient or necessary, as a complement to Boolean networks. We identify and explore the properties of complex subnetworks that are distillable into a single logic relationship. We also identify cyclic subnetworks that ensure the stabilization of the state of participating nodes regardless of the rest of the network. We identify the logic backbone of biomolecular networks, consisting of external signals, self-sustaining cyclic subnetworks (stable motifs), and output nodes. Furthermore, we use the logic framework to identify crucial nodes whose override can drive the system from one steady state to another. We apply these techniques to two biological networks: the epithelial-to-mesenchymal transition network corresponding to a developmental process exploited in tumor invasion, and the network of abscisic acid induced stomatal closure in plants. We find interesting subnetworks with logical implications in these networks. Using these subgraphs and motifs, we efficiently reduce both networks to succinct backbone structures. The logic representation identifies the causal relationships between distant nodes and subnetworks. This knowledge can form the basis of network control or used in the reverse engineering of networks.

Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by 15N NMR relaxation methods

International Nuclear Information System (INIS)

Canales-Mayordomo, Angeles; Fayos, Rosa; Angulo, Jesus; Ojeda, Rafael; Martin-Pastor, Manuel; Nieto, Pedro M.; Martin-Lomas, Manuel; Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus

2006-01-01

The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)

Transforming plastic surfaces with electrophilic backbones from hydrophobic to hydrophilic.

Science.gov (United States)

Kim, Samuel; Bowen, Raffick A R; Zare, Richard N

2015-01-28

We demonstrate a simple nonaqueous reaction scheme for transforming the surface of plastics from hydrophobic to hydrophilic. The chemical modification is achieved by base-catalyzed trans-esterification with polyols. It is permanent, does not release contaminants, and causes no optical or mechanical distortion of the plastic. We present contact angle measurements to show successful modification of several types of plastics including poly(ethylene terephthalate) (PET) and polycarbonate (PC). Its applicability to blood analysis is explored using chemically modified PET blood collection tubes and found to be quite satisfactory. We expect this approach will reduce the cost of manufacturing plastic devices with optimized wettability and can be generalized to other types of plastic materials having an electrophilic linkage as its backbone.

The Role of Templating in the Emergence of RNA from the Prebiotic Chemical Mixture

Directory of Open Access Journals (Sweden)

Andrew S. Tupper

2017-10-01

Full Text Available Biological RNA is a uniform polymer in three senses: it uses nucleotides of a single chirality; it uses only ribose sugars and four nucleobases rather than a mixture of other sugars and bases; and it uses only 3′-5′ bonds rather than a mixture of different bond types. We suppose that prebiotic chemistry would generate a diverse mixture of potential monomers, and that random polymerization would generate non-uniform strands of mixed chirality, monomer composition, and bond type. We ask what factors lead to the emergence of RNA from this mixture. We show that template-directed replication can lead to the emergence of all the uniform properties of RNA by the same mechanism. We study a computational model in which nucleotides react via polymerization, hydrolysis, and template-directed ligation. Uniform strands act as templates for ligation of shorter oligomers of the same type, whereas mixed strands do not act as templates. The three uniform properties emerge naturally when the ligation rate is high. If there is an exact symmetry, as with the chase of chirality, the uniform property arises via a symmetry-breaking phase transition. If there is no exact symmetry, as with monomer selection and backbone regioselectivity, the uniform property emerges gradually as the rate of template-directed ligation is increased.

High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

Directory of Open Access Journals (Sweden)

Kevin A Wilkinson

2008-04-01

Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein

International Nuclear Information System (INIS)

Żerko, Szymon; Byrski, Piotr; Włodarczyk-Pruszyński, Paweł; Górka, Michał; Ledolter, Karin; Masliah, Eliezer; Konrat, Robert; Koźmiński, Wiktor

2016-01-01

New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein

Energy Technology Data Exchange (ETDEWEB)

Żerko, Szymon; Byrski, Piotr; Włodarczyk-Pruszyński, Paweł; Górka, Michał [University of Warsaw, Faculty of Chemistry, Biological and Chemical Research Centre (Poland); Ledolter, Karin [University of Vienna, Department of Computational and Structural Biology, Max F. Perutz Laboratories (Austria); Masliah, Eliezer [University of California, San Diego, Departments of Neuroscience and Pathology (United States); Konrat, Robert [University of Vienna, Department of Computational and Structural Biology, Max F. Perutz Laboratories (Austria); Koźmiński, Wiktor, E-mail: kozmin@chem.uw.edu.pl [University of Warsaw, Faculty of Chemistry, Biological and Chemical Research Centre (Poland)

2016-08-15

New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

Pemetrexed With Platinum Combination as a Backbone for Targeted Therapy in Non-Small-Cell Lung Cancer.

Science.gov (United States)

Stinchcombe, Thomas E; Borghaei, Hossein; Barker, Scott S; Treat, Joseph Anthony; Obasaju, Coleman

2016-01-01

Standard platinum-based chemotherapy combinations for advanced non-small-cell lung cancer (NSCLC) have reached a plateau in terms of the survival benefit they offer for patients. In addition, the emerging clinical trend of tailored treatment based on patient characteristics has led to the development of therapeutic strategies that target specific cancer-related molecular pathways, including epidermal growth factor receptor (EGFR), angiogenesis, and anaplastic lymphoma kinase inhibitors. Current research is focused on combining targeted therapy with platinum-based chemotherapy in an endeavor to achieve an additional benefit in specific patient populations. Currently, pemetrexed is indicated for use in the first-line, maintenance, and second-line settings for the treatment of nonsquamous NSCLC. The combination of pemetrexed and cisplatin is well tolerated and is the approved standard first-line therapy. Thus, the pemetrexed-platinum backbone provides an attractive option for combination with targeted therapies. This review aims to summarize the current knowledge and future prospects of the use of pemetrexed-platinum as a backbone for combination with targeted therapies for NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

TRX-LOGOS - a graphical tool to demonstrate DNA information content dependent upon backbone dynamics in addition to base sequence.

Science.gov (United States)

Fortin, Connor H; Schulze, Katharina V; Babbitt, Gregory A

2015-01-01

It is now widely-accepted that DNA sequences defining DNA-protein interactions functionally depend upon local biophysical features of DNA backbone that are important in defining sites of binding interaction in the genome (e.g. DNA shape, charge and intrinsic dynamics). However, these physical features of DNA polymer are not directly apparent when analyzing and viewing Shannon information content calculated at single nucleobases in a traditional sequence logo plot. Thus, sequence logos plots are severely limited in that they convey no explicit information regarding the structural dynamics of DNA backbone, a feature often critical to binding specificity. We present TRX-LOGOS, an R software package and Perl wrapper code that interfaces the JASPAR database for computational regulatory genomics. TRX-LOGOS extends the traditional sequence logo plot to include Shannon information content calculated with regard to the dinucleotide-based BI-BII conformation shifts in phosphate linkages on the DNA backbone, thereby adding a visual measure of intrinsic DNA flexibility that can be critical for many DNA-protein interactions. TRX-LOGOS is available as an R graphics module offered at both SourceForge and as a download supplement at this journal. To demonstrate the general utility of TRX logo plots, we first calculated the information content for 416 Saccharomyces cerevisiae transcription factor binding sites functionally confirmed in the Yeastract database and matched to previously published yeast genomic alignments. We discovered that flanking regions contain significantly elevated information content at phosphate linkages than can be observed at nucleobases. We also examined broader transcription factor classifications defined by the JASPAR database, and discovered that many general signatures of transcription factor binding are locally more information rich at the level of DNA backbone dynamics than nucleobase sequence. We used TRX-logos in combination with MEGA 6.0 software

Contribution of peptide backbone to Anti-citrulline-dependent antibody reactivity

DEFF Research Database (Denmark)

Trier, Nicole Hartwig; Dam, Catharina; Olsen, Dorthe

2015-01-01

for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone...... found in up to 70% of RA patients’ sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target...... homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g. why some Cit-Gly-containing sequences are not targeted by ACPAs....

Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by {sup 15}N NMR relaxation methods

Energy Technology Data Exchange (ETDEWEB)

Canales-Mayordomo, Angeles; Fayos, Rosa [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain); Angulo, Jesus; Ojeda, Rafael [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Martin-Pastor, Manuel [Unidad de RM y Unidad de RMN de Biomoleculas Asociada al CSIC, Laboratorio de Estructura e Estructura de Biomoleculas Jose Carracido (Spain); Nieto, Pedro M.; Martin-Lomas, Manuel [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain)], E-mail: jjbarbero@cib.csic.es

2006-08-15

The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)

On the purported "backbone fluorescence" in protein three-dimensional fluorescence spectra

DEFF Research Database (Denmark)

Bortolotti, Annalisa; Wong, Yin How; Korsholm, Stine S.

2016-01-01

In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220-230 nm. In the last 10 years we have observed a worrying increase in the number of articles...... as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence...... as an established fact. We hope the current paper helps counter this new situation and leads to a reassessment of those papers that make this erroneous claim....

Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data

Science.gov (United States)

Hamuro, Yoshitomo; E, Sook Yen

2018-05-01

The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. [Figure not available: see fulltext.

Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data.

Science.gov (United States)

Hamuro, Yoshitomo; E, Sook Yen

2018-05-01

The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. Graphical Abstract ᅟ.

Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data

Science.gov (United States)

Hamuro, Yoshitomo; E, Sook Yen

2018-03-01

The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. [Figure not available: see fulltext.

Expansion of the known Klebsiella pneumoniae species gene pool by characterization of novel alien DNA islands integrated into tmRNA gene sites.

Science.gov (United States)

Zhang, Jie; van Aartsen, Jon Jurriaan; Jiang, Xiaofei; Shao, Yucheng; Tai, Cui; He, Xinyi; Tan, Zhilei; Deng, Zixin; Jia, Shiru; Rajakumar, Kumar; Ou, Hong-Yu

2011-02-01

Klebsiella pneumoniae is an important bacterial pathogen of man that is commonly associated with opportunistic and hospital-associated infections. Increasing levels of multiple-antibiotic resistance associated with this species pose a major emerging clinical problem. This organism also occurs naturally in other diverse environments, including the soil. Consistent with its varied lifestyle and membership of the Enterobacteriaceae family, K. pneumoniae genomes exhibit highly plastic architecture comprising a core genome backbone interspersed with numerous and varied alien genomic islands. In this study the size of the presently known K. pneumoniae pan-genome gene pool was estimated through analysis of complete sequences of three chromosomes and 31 plasmids belonging to K. pneumoniae strains. In addition, using a PCR-based strategy the genomic content of eight tRNA/tmRNA gene sites that serve as DNA insertion hotspots were investigated in 28 diverse environmental and clinical strains of K. pneumoniae. Sequencing and characterization of five newly identified horizontally-acquired tmRNA-associated islands further expanded the archived K. pneumoniae gene pool to a total of 7648 unique gene members. Large-scale investigation of the content of tRNA/tmRNA hotspots will be useful to identify and/or survey accessory sequences dispersed amongst hundreds to thousands of members of many key bacterial species. Copyright © 2010 Elsevier B.V. All rights reserved.

An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.

Directory of Open Access Journals (Sweden)

Glen John McIntyre

Full Text Available Vector construction with restriction enzymes (REs typically involves the ligation of a digested donor fragment (insert to a reciprocally digested recipient fragment (vector backbone. Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA expression vectors for RNA interference (RNAi studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.

TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts

Energy Technology Data Exchange (ETDEWEB)

Shen Yang; Delaglio, Frank [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Cornilescu, Gabriel [National Magnetic Resonance Facility (United States); Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: bax@nih.gov

2009-08-15

NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between {sup 13}C, {sup 15}N and {sup 1}H chemical shifts and backbone torsion angles {phi} and {psi} (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted {phi} and {psi} angles, equals {+-}13{sup o}. Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C′ chemical shifts of multiple contiguous residues in highly resolved 3D spectra

International Nuclear Information System (INIS)

Yoshimura, Yuichi; Kulminskaya, Natalia V.; Mulder, Frans A. A.

2015-01-01

Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone 1 H N , 15 N H , and 13 C′ resonance assignments to be completed from a single pair of 3D experiments

A Bayesian-probability-based method for assigning protein backbone dihedral angles based on chemical shifts and local sequences

Energy Technology Data Exchange (ETDEWEB)

Wang Jun; Liu Haiyan [University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at the Microscale, and Key Laboratory of Structural Biology, School of Life Sciences (China)], E-mail: hyliu@ustc.edu.cn

2007-01-15

Chemical shifts contain substantial information about protein local conformations. We present a method to assign individual protein backbone dihedral angles into specific regions on the Ramachandran map based on the amino acid sequences and the chemical shifts of backbone atoms of tripeptide segments. The method uses a scoring function derived from the Bayesian probability for the central residue of a query tripeptide segment to have a particular conformation. The Ramachandran map is partitioned into representative regions at two levels of resolution. The lower resolution partitioning is equivalent to the conventional definitions of different secondary structure regions on the map. At the higher resolution level, the {alpha} and {beta} regions are further divided into subregions. Predictions are attempted at both levels of resolution. We compared our method with TALOS using the original TALOS database, and obtained comparable results. Although TALOS may produce the best results with currently available databases which are much enlarged, the Bayesian-probability-based approach can provide a quantitative measure for the reliability of predictions.

Sequential backbone assignment based on dipolar amide-to-amide correlation experiments

Energy Technology Data Exchange (ETDEWEB)

Xiang, ShengQi; Grohe, Kristof; Rovó, Petra; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus, E-mail: rali@nmr.mpibpc.mpg.de [Max Planck Institute for Biophysical Chemistry, Department for NMR-Based Structural Biology (Germany)

2015-07-15

Proton detection in solid-state NMR has seen a tremendous increase in popularity in the last years. New experimental techniques allow to exploit protons as an additional source of information on structure, dynamics, and protein interactions with their surroundings. In addition, sensitivity is mostly improved and ambiguity in assignment experiments reduced. We show here that, in the solid state, sequential amide-to-amide correlations turn out to be an excellent, complementary way to exploit amide shifts for unambiguous backbone assignment. For a general assessment, we compare amide-to-amide experiments with the more common {sup 13}C-shift-based methods. Exploiting efficient CP magnetization transfers rather than less efficient INEPT periods, our results suggest that the approach is very feasible for solid-state NMR.

Sequential backbone assignment based on dipolar amide-to-amide correlation experiments

International Nuclear Information System (INIS)

Xiang, ShengQi; Grohe, Kristof; Rovó, Petra; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus

2015-01-01

Proton detection in solid-state NMR has seen a tremendous increase in popularity in the last years. New experimental techniques allow to exploit protons as an additional source of information on structure, dynamics, and protein interactions with their surroundings. In addition, sensitivity is mostly improved and ambiguity in assignment experiments reduced. We show here that, in the solid state, sequential amide-to-amide correlations turn out to be an excellent, complementary way to exploit amide shifts for unambiguous backbone assignment. For a general assessment, we compare amide-to-amide experiments with the more common 13 C-shift-based methods. Exploiting efficient CP magnetization transfers rather than less efficient INEPT periods, our results suggest that the approach is very feasible for solid-state NMR

Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

OpenAIRE

Nougairede, Antoine; Klitting, Raphaelle; Aubry, Fabien; Gilles, Magali; Touret, Franck; De Lamballerie, Xavier

2018-01-01

Zika virus (ZIKV) recently dispersed throughout the tropics and sub-tropics causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. Here we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared diff...

Phosphodiester content measured in human liver by in vivo 31 P MR spectroscopy at 7 tesla.

Science.gov (United States)

Purvis, Lucian A B; Clarke, William T; Valkovič, Ladislav; Levick, Christina; Pavlides, Michael; Barnes, Eleanor; Cobbold, Jeremy F; Robson, Matthew D; Rodgers, Christopher T

2017-12-01

Phosphorus ( 31 P) metabolites are emerging liver disease biomarkers. Of particular interest are phosphomonoester and phosphodiester (PDE) "peaks" that comprise multiple overlapping resonances in 31 P spectra. This study investigates the effect of improved spectral resolution at 7 Tesla (T) on quantifying hepatic metabolites in cirrhosis. Five volunteers were scanned to determine metabolite T 1 s. Ten volunteers and 11 patients with liver cirrhosis were scanned at 7T. Liver spectra were acquired in 28 min using a 16-channel 31 P array and 3D chemical shift imaging. Concentrations were calculated using γ-adenosine-triphosphate (γ-ATP) = 2.65 mmol/L wet tissue. T 1 means ± standard deviations: phosphatidylcholine 1.05 ± 0.28 s, nicotinamide-adenine-dinucleotide (NAD + ) 2.0 ± 1.0 s, uridine-diphosphoglucose (UDPG) 3.3 ± 1.4 s. Concentrations in healthy volunteers: α-ATP 2.74 ± 0.11 mmol/L wet tissue, inorganic phosphate 2.23 ± 0.20 mmol/L wet tissue, glycerophosphocholine 2.34 ± 0.46 mmol/L wet tissue, glycerophosphoethanolamine 1.50 ± 0.28 mmol/L wet tissue, phosphocholine 1.06 ± 0.16 mmol/L wet tissue, phosphoethanolamine 0.77 ± 0.14 mmol/L wet tissue, NAD + 2.37 ± 0.14 mmol/L wet tissue, UDPG 2.00 ± 0.22 mmol/L wet tissue, phosphatidylcholine 1.38 ± 0.31 mmol/L wet tissue. Inorganic phosphate and phosphatidylcholine concentrations were significantly lower in patients; glycerophosphoethanolamine concentrations were significantly higher (P < 0.05). We report human in vivo hepatic T 1 s for phosphatidylcholine, NAD + , and UDPG for the first time at 7T. Our protocol allows high signal-to-noise, repeatable measurement of metabolite concentrations in human liver. The splitting of PDE into its constituent peaks at 7T may allow more insight into changes in metabolism. Magn Reson Med 78:2095-2105, 2017. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on

Effects of a vanadium post-metallocene catalyst-induced polymer backbone inhomogeneity on UV oxidative degradation of the resulting polyethylene film

KAUST Repository

Atiqullah, M.; Winston, M. S.; Bercaw, J. E.; Hussain, I.; Fazal, A.; Al-Harthi, M. A.; Emwas, A. H M; Khan, M. J.; Hossaen, A.

2012-01-01

(nm-CopolyPE) with 1-hexene having very low backbone unsaturation. The nm-CopolyPE inhomogeneity was reflected in the distributions of short chain branches, 1-hexene composition, and methylene sequence length. The 1-hexene incorporation

Synthesis of branched–backbone oligosaccharides of the pectic RG-I plant cell wall polysaccharide

DEFF Research Database (Denmark)

Awan, Shahid Iqbal; Clausen, Mads Hartvig

with numerous branches of galactan, arabinan, or arabinogalactan positioned at C-4 of the rhamnose residues. The use of defined oligosaccharides rather than isolated polysaccharides can aid in obtaining detailedinformation about biosynthetic pathways, plant evolution, and agronomical properties. Furthermore......,biological testing can provide new insight into plant biology; important for plant preservation, engineering,and utilization of plants as a source of bioenergy. Present work towards defined RG-I substructures involvesa [4+3]-coupling to furnish a heptasaccharide backbone unit (see Figure 1). Moreover, installation...

Investigation of novel solid oxide fuel cell cathodes based on impregnation of SrTixFe1-xO3-δ into ceria-based backbones

DEFF Research Database (Denmark)

Brinch-Larsen, Mathias; Søgaard, Martin; Hjelm, Johan

2013-01-01

Solid oxide fuel cell (SOFC) cathodes were prepared by impregnating the nitrates corresponding to SrTixFe1-xO3-δ (STF), x= 0; 0.1; 0.2; 0.3; 0.4 and 0.5, into a porous backbone of Ce 0.9Gd0.1O2-δ (CGO). STF was chosen as very high oxygen surface exchange rate, high ionic conductivity and electroc......Solid oxide fuel cell (SOFC) cathodes were prepared by impregnating the nitrates corresponding to SrTixFe1-xO3-δ (STF), x= 0; 0.1; 0.2; 0.3; 0.4 and 0.5, into a porous backbone of Ce 0.9Gd0.1O2-δ (CGO). STF was chosen as very high oxygen surface exchange rate, high ionic conductivity...... backbone. All prepared electrodes were characterized as symmetric cells using impedance spectroscopy. Within the investigated series the infiltrate with x = 0.1 (STF10) showed the best performance with an area specific resistance (ASR) of ASR ≈ 6.4 Ω cm2 (STF10) at 600°C in air. The relatively poor...

Thermoresponsive Poly(2-oxazoline) Molecular Brushes by Living Ionic Polymerization: Kinetic Investigations of Pendant Chain Grafting and Cloud Point Modulation by Backbone and Side Chain Length Variation

KAUST Repository

Zhang, Ning

2012-04-17

Molecular brushes of poly(2-oxazoline)s were prepared by living anionic polymerization of 2-iso-propenyl-2-oxazoline to form the backbone and subsequent living cationic ring-opening polymerization of 2-n- or 2-iso-propyl-2-oxazoline for pendant chain grafting. In situ kinetic studies indicate that the initiation efficiency and polymerization rates are independent from the number of initiator functions per initiator molecule. This was attributed to the high efficiency of oxazolinium salt and the stretched conformation of the backbone, which is caused by the electrostatic repulsion of the oxazolinium moieties along the macroinitiator. The resulting molecular brushes showed thermoresponsive properties, that is, having a defined cloud point (CP). The dependence of the CP as a function of backbone and side chain length as well as concentration was studied. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trappist: european project dedicated to an open backbone structure for NDT expertise

International Nuclear Information System (INIS)

Nouailhas, B.; Vailhen, O.

1993-01-01

Non Destructive Testing (NDT) on critical components such as the reactor vessel, primary coolant pipes and steam generators have already been, and are still the subject of many development concerning the improvement of measuring techniques, data processing and on site operation. The tools developed for these tests are generally closed, difficult to extend and of proprietary type. Productivity could be increased if an open backbone structure common to several types of test were available. Moreover, these components are generally submitted to a test involving a single method. In certain cases, the produced information is an insufficient basis for drawing up a satisfactory diagnosis: the test operator or expert often faces problems in extracting more information from signals that are generally noisy. It may prove necessary to complete the inspection with another NDT method based on different principles in order to obtain better performances. It is then by combining the information obtained by two complementary methods that it will be possible to draw up a more reliable diagnosis. These components have also a complex shape. In the case of ultrasonic testing, the accurate following of probe paths requires 3D representation of the geometry, as it is built, to position and display the data obtained from the inspection. To take these geometric constraints into account, it is imperative to use computer tools allowing the three-dimensional representation of the reconstructed information on the components' actual geometry. This specific difficulty, which has long been appreciated, is the subject of developments resulting to industrial products that are more or less satisfactory. The aim of the European Project TRAPPIST (Race Program) is to study an open backbone structure. A mock-up of an analysis station dedicated to NDT expertise will be built and evaluated with specific examples. (authors). 6 figs., 1 ref

40-Gbps optical backbone network deep packet inspection based on FPGA

Science.gov (United States)

Zuo, Yuan; Huang, Zhiping; Su, Shaojing

2014-11-01

In the era of information, the big data, which contains huge information, brings about some problems, such as high speed transmission, storage and real-time analysis and process. As the important media for data transmission, the Internet is the significant part for big data processing research. With the large-scale usage of the Internet, the data streaming of network is increasing rapidly. The speed level in the main fiber optic communication of the present has reached 40Gbps, even 100Gbps, therefore data on the optical backbone network shows some features of massive data. Generally, data services are provided via IP packets on the optical backbone network, which is constituted with SDH (Synchronous Digital Hierarchy). Hence this method that IP packets are directly mapped into SDH payload is named POS (Packet over SDH) technology. Aiming at the problems of real time process of high speed massive data, this paper designs a process system platform based on ATCA for 40Gbps POS signal data stream recognition and packet content capture, which employs the FPGA as the CPU. This platform offers pre-processing of clustering algorithms, service traffic identification and data mining for the following big data storage and analysis with high efficiency. Also, the operational procedure is proposed in this paper. Four channels of 10Gbps POS signal decomposed by the analysis module, which chooses FPGA as the kernel, are inputted to the flow classification module and the pattern matching component based on TCAM. Based on the properties of the length of payload and net flows, buffer management is added to the platform to keep the key flow information. According to data stream analysis, DPI (deep packet inspection) and flow balance distribute, the signal is transmitted to the backend machine through the giga Ethernet ports on back board. Practice shows that the proposed platform is superior to the traditional applications based on ASIC and NP.

Control of polymer-packing orientation in thin films through synthetic tailoring of backbone coplanarity

KAUST Repository

Chen, Mark S.

2013-10-22

Controlling solid-state order of π-conjugated polymers through macromolecular design is essential for achieving high electronic device performance; yet, it remains a challenge, especially with respect to polymer-packing orientation. Our work investigates the influence of backbone coplanarity on a polymer\\'s preference to pack face-on or edge-on relative to the substrate. Isoindigo-based polymers were synthesized with increasing planarity by systematically substituting thiophenes for phenyl rings in the acceptor comonomer. This increasing backbone coplanarity, supported by density functional theory (DFT) calculations of representative trimers, leads to the narrowing of polymer band gaps as characterized by ultraviolet-visible-near infrared (UV-vis-NIR) spectroscopy and cyclic voltammetry. Among the polymers studied, regiosymmetric II and TII polymers exhibited the highest hole mobilities in organic field-effect transistors (OFETs), while in organic photovoltaics (OPVs), TBII polymers that display intermediate levels of planarity provided the highest power conversion efficiencies. Upon thin-film analysis by atomic force microscropy (AFM) and grazing-incidence X-ray diffraction (GIXD), we discovered that polymer-packing orientation could be controlled by tuning polymer planarity and solubility. Highly soluble, planar polymers favor face-on orientation in thin films while the less soluble, nonplanar polymers favor an edge-on orientation. This study advances our fundamental understanding of how polymer structure influences nanostructural order and reveals a new synthetic strategy for the design of semiconducting materials with rationally engineered solid-state properties. © 2013 American Chemical Society.

Backbone dynamics of reduced plastocyanin from the cyanobacterium Anabaena variabilis: Regions involved in electron transfer have enhanced mobility

DEFF Research Database (Denmark)

Ma, L.X.; Hass, M.A.S.; Vierick, N.

2003-01-01

The dynamics of the backbone of the electron-transfer protein plastocyanin from the cyanobacterium Anabaena variabilis were determined from the N-15 and C-13(alpha) R-1 and R-2) relaxation rates and steady-state [H-1]-N-15 and [H-1]-C-13 nuclear Overhauser effects (NOEs) using the model...

Data on the effect of in vivo knockdown using artificial ErbB3 miRNA on Remak bundle structure

Directory of Open Access Journals (Sweden)

Yuki Miyamoto

2017-06-01

Full Text Available Mature Schwann cells, the peripheral nervous system (PNS glial cells, have two major roles for neuronal axons (Bunge, 1993 [1]. For large diameter axons, Schwann cells form myelin sheaths with multiple layers. For small diameter axons, they form Remak bundle composed only of single layer of the Schwann cell plasma membrane. In the PNS, ErbB3 forms a dimer with ErbB2 on the Schwann cell plasma membrane. ErbB3 plays a key role in myelination by myelinating Schwann cells, that is to say, its role in myelin thickness. Herein we provide the data regarding the effect of in vivo knockdown of ErbB3 on the thickness between an axon and a neighboring axon in Remak bundle, which is formed by non-myelinating Schwann cells. Since ErbB3 knockout mice are embryonically lethal, Schwann cell lineage-specific transgenic mice transcribing ErbB3 shRNA with an artificial miRNA backbone were generated and used in these experiments (Torii et al., 2014 [2].

Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology

International Nuclear Information System (INIS)

Shen Yang; Bax, Ad

2007-01-01

Chemical shifts of nuclei in or attached to a protein backbone are exquisitely sensitive to their local environment. A computer program, SPARTA, is described that uses this correlation with local structure to predict protein backbone chemical shifts, given an input three-dimensional structure, by searching a newly generated database for triplets of adjacent residues that provide the best match in φ/ψ/χ 1 torsion angles and sequence similarity to the query triplet of interest. The database contains 15 N, 1 H N , 1 H α , 13 C α , 13 C β and 13 C' chemical shifts for 200 proteins for which a high resolution X-ray (≤2.4 A) structure is available. The relative importance of the weighting factors for the φ/ψ/χ 1 angles and sequence similarity was optimized empirically. The weighted, average secondary shifts of the central residues in the 20 best-matching triplets, after inclusion of nearest neighbor, ring current, and hydrogen bonding effects, are used to predict chemical shifts for the protein of known structure. Validation shows good agreement between the SPARTA-predicted and experimental shifts, with standard deviations of 2.52, 0.51, 0.27, 0.98, 1.07 and 1.08 ppm for 15 N, 1 H N , 1 H α , 13 C α , 13 C β and 13 C', respectively, including outliers

Free vibration analysis of a robotic fish based on a continuous and non-uniform flexible backbone with distributed masses

Science.gov (United States)

Coral, W.; Rossi, C.; Curet, O. M.

2015-12-01

This paper presents a Differential Quadrature Element Method for free transverse vibration of a robotic fish based on a continuous and non-uniform flexible backbone with distributed masses (fish ribs). The proposed method is based on the theory of a Timoshenko cantilever beam. The effects of the masses (number, magnitude and position) on the value of natural frequencies are investigated. Governing equations, compatibility and boundary conditions are formulated according to the Differential Quadrature rules. The convergence, efficiency and accuracy are compared to other analytical solution proposed in the literature. Moreover, the proposed method has been validate against the physical prototype of a flexible fish backbone. The main advantages of this method, compared to the exact solutions available in the literature are twofold: first, smaller computational cost and second, it allows analysing the free vibration in beams whose section is an arbitrary function, which is normally difficult or even impossible with other analytical methods.

(nBuCp)2ZrCl2-catalyzed Ethylene-4M1P Copolymerization: Copolymer Backbone Structure, Melt Behavior, and Crystallization

KAUST Repository

Atiqullah, Muhammad; Adamu, Sagir; Malaibari, Zuhair O.; Al-Harthi, Mamdouh A.; Emwas, Abdul-Hamid M.

2016-01-01

The judicious design of methylaluminoxane (MAO) anions expands the scope for developing industrial metallocene catalysts. Therefore, the effects of MAO anion design on the backbone structure, melt behavior, and crystallization of ethylene−4-methyl-1

Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

Directory of Open Access Journals (Sweden)

Hiraku Takada

Full Text Available Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon, within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons

Remote consultation and diagnosis in medical imaging using a global PACS backbone network

Science.gov (United States)

Martinez, Ralph; Sutaria, Bijal N.; Kim, Jinman; Nam, Jiseung

1993-10-01

A Global PACS is a national network which interconnects several PACS networks at medical and hospital complexes using a national backbone network. A Global PACS environment enables new and beneficial operations between radiologists and physicians, when they are located in different geographical locations. One operation allows the radiologist to view the same image folder at both Local and Remote sites so that a diagnosis can be performed. The paper describes the user interface, database management, and network communication software which has been developed in the Computer Engineering Research Laboratory and Radiology Research Laboratory. Specifically, a design for a file management system in a distributed environment is presented. In the remote consultation and diagnosis operation, a set of images is requested from the database archive system and sent to the Local and Remote workstation sites on the Global PACS network. Viewing the same images, the radiologists use pointing overlay commands, or frames to point out features on the images. Each workstation transfers these frames, to the other workstation, so that an interactive session for diagnosis takes place. In this phase, we use fixed frames and variable size frames, used to outline an object. The data pockets for these frames traverses the national backbone in real-time. We accomplish this feature by using TCP/IP protocol sockets for communications. The remote consultation and diagnosis operation has been tested in real-time between the University Medical Center and the Bowman Gray School of Medicine at Wake Forest University, over the Internet. In this paper, we show the feasibility of the operation in a Global PACS environment. Future improvements to the system will include real-time voice and interactive compressed video scenarios.

The RNA gene information: retroelement-microRNA entangling as the RNA quantum code.

Science.gov (United States)

Fujii, Yoichi Robertus

2013-01-01

MicroRNA (miRNA) and retroelements may be a master of regulator in our life, which are evolutionally involved in the origin of species. To support the Darwinism from the aspect of molecular evolution process, it has tremendously been interested in the molecular information of naive RNA. The RNA wave model 2000 consists of four concepts that have altered from original idea of the miRNA genes for crosstalk among embryonic stem cells, their niche cells, and retroelements as a carrier vesicle of the RNA genes. (1) the miRNA gene as a mobile genetic element induces transcriptional and posttranscriptional silencing via networking-processes (no hierarchical architecture); (2) the RNA information supplied by the miRNA genes expands to intracellular, intercellular, intraorgan, interorgan, intraspecies, and interspecies under the cycle of life into the global environment; (3) the mobile miRNAs can self-proliferate; and (4) cells contain two types information as resident and genomic miRNAs. Based on RNA wave, we have developed an interest in investigation of the transformation from RNA information to quantum bits as physicochemical characters of RNA with the measurement of RNA electron spin. When it would have been given that the fundamental bases for the acquired characters in genetics can be controlled by RNA gene information, it may be available to apply for challenging against RNA gene diseases, such as stress-induced diseases.

Effects of a vanadium post-metallocene catalyst-induced polymer backbone inhomogeneity on UV oxidative degradation of the resulting polyethylene film

KAUST Repository

Atiqullah, M.

2012-07-01

A Group 5 post-metallocene precatalyst, (ONO)VCl(THF) 2 (ONO = a bis(phenolate)pyridine LX 2 pincer ligand), activated with modified methylaluminoxane (MMAO-3A) produced a linear ethylene homopolymer (nm-HomoPE)and an unusual inhomogeneous copolymer (nm-CopolyPE) with 1-hexene having very low backbone unsaturation. The nm-CopolyPE inhomogeneity was reflected in the distributions of short chain branches, 1-hexene composition, and methylene sequence length. The 1-hexene incorporation into the polyethylene backbone strongly depended on the molecular weight of the growing polymer chain. (ONO)VCl(THF) 2, because of site diversity and easier removal of a tertiary (vs. a secondary) hydrogen, produced a skewed short chain branching (SCB) profile, incorporating 1-hexene more efficiently in the low molecular weight region than in the high molecular weight region. The significant decrease in molecular weight by 1-hexene showed that the (ONO)VCl(THF) 2 catalytic sites were also highly responsive to chain-transfer directly to 1-hexene itself, producing vinyl and trans-vinylene termini. Subsequently, the effect of backbone inhomogeneity on the UV oxidative degradation of films made from both polyethylenes was investigated. The major functional group accumulated in the branched nm-CopolyPE film was carbonyl followed by carboxyl, then vinyl/ester, whereas that in the linear nm-HomoPE film was carboxyl. However, (carbonyl, carboxyl, vinyl, and ester) nm-CopolyPE film >> (carboxyl) nm-HomoPE film). The distributions of the tertiary C-H sites and methylene sequence length in the branched nm-CopolyPE film enhanced abstraction of H, decomposition of hydroperoxide group ROOH, and generation of carbonyl compounds as compared with those in the linear nm-HomoPE film. This clearly establishes the role played by the backbone inhomogeneity. The effect of short chain branches and sequence length distributions on peak melting temperature T pm, and most probably lamellar thickness L o, was

The extension of a DNA double helix by an additional Watson-Crick base pair on the same backbone.

Science.gov (United States)

Kumar, Pawan; Sharma, Pawan K; Madsen, Charlotte S; Petersen, Michael; Nielsen, Poul

2013-06-17

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solution structure of tRNA{sup Val} from refinement of homology model against residual dipolar coupling and SAXS data

Energy Technology Data Exchange (ETDEWEB)

Grishaev, Alexander, E-mail: AlexanderG@intra.niddk.nih.gov; Ying, Jinfa [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Canny, Marella D.; Pardi, Arthur [University of Colorado, Boulder, Department of Chemistry and Biochemistry, 215 UCB (United States)], E-mail: Arthur.Pardi@Colorado.edu; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: bax@nih.gov

2008-10-15

A procedure is presented for refinement of a homology model of E. coli tRNA{sup Val}, originally based on the X-ray structure of yeast tRNA{sup Phe}, using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-H{sup N} RDCs measured with Pf1 phage alignment, and 20 imino N-H{sup N} RDCs obtained from magnetic field dependent alignment of tRNA{sup Val}. The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA{sup Phe} by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q{sub free} = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA{sup Phe}, in agreement with previous NMR-based tRNA{sup Val} models.

Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

International Nuclear Information System (INIS)

Sahu, Sarata C.; Bhuyan, Abani K.; Udgaonkar, Jayant B.; Hosur, R.V.

2000-01-01

Backbone dynamics of uniformly 15 N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 deg. C, pH 6.6, using 15 N relaxation data obtained from proton-detected 2D { 1 H}- 15 N NMR spectroscopy. 15 N spin-lattice relaxation rate constants (R 1 ), spin-spin relaxation rate constants (R 2 ), and steady-state heteronuclear { 1 H}- 15 N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide 15 N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (τ m ) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme

Controlled Conjugated Backbone Twisting for an Increased Open-Circuit Voltage while Having a High Short-Circuit Current in Poly(hexylthiophene) Derivatives

KAUST Repository

Ko, Sangwon; Hoke, Eric T.; Pandey, Laxman; Hong, Sanghyun; Mondal, Rajib; Risko, Chad; Yi, Yuanping; Noriega, Rodrigo; McGehee, Michael D.; Bré das, Jean-Luc; Salleo, Alberto; Bao, Zhenan

2012-01-01

and charge-transfer state energy of the polymer:fullerene BHJ solar cells increased substantially with the degree of twist induced within the conjugated backbone-due to an increase in the polymer ionization potential-while the short-circuit current decreased

Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

Directory of Open Access Journals (Sweden)

Shi Qun Zhang

2016-05-01

Full Text Available The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3-glucan, a crucial pathogen-associated molecular pattern (PAMP of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.

iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

Directory of Open Access Journals (Sweden)

Jittrawan Thaiprasit

2016-03-01

Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

Exploring the molecular basis of dsRNA recognition by NS1 protein of influenza A virus using molecular dynamics simulation and free energy calculation.

Science.gov (United States)

Pan, Dabo; Sun, Huijun; Shen, Yulin; Liu, Huanxiang; Yao, Xiaojun

2011-12-01

The frequent outbreak of influenza pandemic and the limited available anti-influenza drugs highlight the urgent need for the development of new antiviral drugs. The dsRNA-binding surface of nonstructural protein 1 of influenza A virus (NS1A) is a promising target. The detailed understanding of NS1A-dsRNA interaction will be valuable for structure-based anti-influenza drug discovery. To characterize and explore the key interaction features between dsRNA and NS1A, molecular dynamics simulation combined with MM-GBSA calculations were performed. Based on the MM-GBSA calculations, we find that the intermolecular van der Waals interaction and the nonpolar solvation term provide the main driving force for the binding process. Meanwhile, 17 key residues from NS1A were identified to be responsible for the dsRNA binding. Compared with the wild type NS1A, all the studied mutants S42A, T49A, R38A, R35AR46A have obvious reduced binding free energies with dsRNA reflecting in the reduction of the polar and/or nonpolar interactions. In addition, the structural and energy analysis indicate the mutations have a small effect to the backbone structures but the loss of side chain interactions is responsible for the decrease of the binding affinity. The uncovering of NS1A-dsRNA recognition mechanism will provide some useful insights and new chances for the development of anti-influenza drugs. Copyright © 2011 Elsevier B.V. All rights reserved.

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...

Data Acquisition Backbone Core DABC release v1.0

International Nuclear Information System (INIS)

Adamczewski-Musch, J; Kurz, N; Linev, S; Essel, H G

2010-01-01

The Data Acquisition Backbone Core (DABC) is a general purpose software framework designed for the implementation of a wide-range of data acquisition systems - from various small detector test beds to high performance systems. DABC consists of a compact data-flow kernel and a number of plug-ins for various functional components like data inputs, device drivers, user functional modules and applications. DABC provides configurable components for implementing event building over fast networks like InfiniBand or Gigabit Ethernet. A generic Java GUI provides the dynamic control and visualization of control parameters and commands, provided by DIM servers. A first set of application plug-ins has been implemented to use DABC as event builder for the front-end components of the GSI standard DAQ system MBS (Multi Branch System). Another application covers the connection to DAQ readout chains from detector front-end boards (N-XYTER) linked to read-out controller boards (ROC) over UDP into DABC for event building, archiving and data serving. This was applied for data taking in the September 2008 test beamtime for the CBM experiment at GSI. DABC version 1.0 is released and available from the website.

RNA-SSPT: RNA Secondary Structure Prediction Tools.

Science.gov (United States)

Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

2013-01-01

The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.

Combinatorics of RNA-RNA interaction

DEFF Research Database (Denmark)

Li, Thomas J X; Reidys, Christian

2012-01-01

RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

Side chain and backbone contributions of Phe508 to CFTR folding

Energy Technology Data Exchange (ETDEWEB)

Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J. (U. of Texas-SMED)

2010-12-07

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

Cross-correlated relaxation rates between protein backbone H–X dipolar interactions

International Nuclear Information System (INIS)

Vögeli, Beat

2017-01-01

The relaxation interference between dipole–dipole interactions of two separate spin pairs carries structural and dynamics information. In particular, when compared to individual dynamic behavior of those spin pairs, such cross-correlated relaxation (CCR) rates report on the correlation between the spin pairs. We have recently mapped out correlated motion along the backbone of the protein GB3, using CCR rates among and between consecutive H N –N and H α –C α dipole–dipole interactions. Here, we provide a detailed account of the measurement of the four types of CCR rates. All rates were obtained from at least two different pulse sequences, of which the yet unpublished ones are presented. Detailed comparisons between the different methods and corrections for unwanted pathways demonstrate that the averaged CCR rates are highly accurate and precise with errors of 1.5–3% of the entire value ranges.

Cross-correlated relaxation rates between protein backbone H–X dipolar interactions

Energy Technology Data Exchange (ETDEWEB)

Vögeli, Beat, E-mail: beat.vogeli@ucdenver.edu [University of Colorado Denver, Department of Biochemistry and Molecular Genetics (United States)

2017-03-15

The relaxation interference between dipole–dipole interactions of two separate spin pairs carries structural and dynamics information. In particular, when compared to individual dynamic behavior of those spin pairs, such cross-correlated relaxation (CCR) rates report on the correlation between the spin pairs. We have recently mapped out correlated motion along the backbone of the protein GB3, using CCR rates among and between consecutive H{sup N}–N and H{sup α}–C{sup α} dipole–dipole interactions. Here, we provide a detailed account of the measurement of the four types of CCR rates. All rates were obtained from at least two different pulse sequences, of which the yet unpublished ones are presented. Detailed comparisons between the different methods and corrections for unwanted pathways demonstrate that the averaged CCR rates are highly accurate and precise with errors of 1.5–3% of the entire value ranges.

Peptide nucleic acids and their potential applications in biotechnology

DEFF Research Database (Denmark)

Buchardt, O.; Egholm, M.; Berg, R.H.

1993-01-01

Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids1-3. PNAs exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They,are resistant to nuclease...

Capturing Snapshots of APE1 Processing DNA Damage

Science.gov (United States)

Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

2015-01-01

DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

Dynamic Resource Allocation and QoS Control Capabilities of the Japanese Academic Backbone Network

Directory of Open Access Journals (Sweden)

Michihiro Aoki

2010-08-01

Full Text Available Dynamic resource control capabilities have become increasingly important for academic networks that must support big scientific research projects at the same time as less data intensive research and educational activities. This paper describes the dynamic resource allocation and QoS control capabilities of the Japanese academic backbone network, called SINET3, which supports a variety of academic applications with a wide range of network services. The article describes the network architecture, networking technologies, resource allocation, QoS control, and layer-1 bandwidth on-demand services. It also details typical services developed for scientific research, including the user interface, resource control, and management functions, and includes performance evaluations.

Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

KAUST Repository

Rydberg, Hanna A.; Matson, Maria; Å mand, Helene L.; Esbjö rner, Elin K.; Nordé n, Bengt

2012-01-01

Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view. © 2012 American Chemical Society.

Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

KAUST Repository

Rydberg, Hanna A.

2012-07-10

Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view. © 2012 American Chemical Society.

Comprehensive characterization of lncRNA-mRNA related ceRNA network across 12 major cancers

Science.gov (United States)

Feng, Li; Li, Feng; Sun, Zeguo; Wu, Tan; Shi, Xinrui; Li, Jing; Li, Xia

2016-01-01

Recent studies indicate that long noncoding RNAs (lncRNAs) can act as competing endogenous RNAs (ceRNAs) to indirectly regulate mRNAs through shared microRNAs, which represents a novel layer of RNA crosstalk and plays critical roles in the development of tumor. However, the global regulation landscape and characterization of these lncRNA related ceRNA crosstalk in cancers is still largely unknown. Here, we systematically characterized the lncRNA related ceRNA interactions across 12 major cancers and the normal physiological states by integrating multidimensional molecule profiles of more than 5000 samples. Our study suggest the large difference of ceRNA regulation between normal and tumor states and the higher similarity across similar tissue origin of tumors. The ceRNA related molecules have more conserved features in tumor networks and they play critical roles in both the normal and tumorigenesis processes. Besides, lncRNAs in the pan-cancer ceRNA network may be potential biomarkers of tumor. By exploring hub lncRNAs, we found that these conserved key lncRNAs dominate variable tumor hallmark processes across pan-cancers. Network dynamic analysis highlights the critical roles of ceRNA regulation in tumorigenesis. By analyzing conserved ceRNA interactions, we found that miRNA mediate ceRNA regulation showed different patterns across pan-cancer; while analyzing the cancer specific ceRNA interactions reveal that lncRNAs synergistically regulated tumor driver genes of cancer hallmarks. Finally, we found that ceRNA modules have the potential to predict patient survival. Overall, our study systematically dissected the lncRNA related ceRNA networks in pan-cancer that shed new light on understanding the molecular mechanism of tumorigenesis. PMID:27580177

Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

Science.gov (United States)

Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

2017-01-01

microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

Characterization of a parallel-stranded DNA hairpin

International Nuclear Information System (INIS)

Germann, M.W.; Vogel, H.J.; Pon, R.T.; van de Sande, J.H.

1989-01-01

Recently, the authors have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure. In the present study, they have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T) 8 C 4 (A) 8 -3' and the related antiparallel (APS) hair 5'-d(T) 8 C 4 (A) 8 -3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31 P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. On the basis of NOESY measurements, they find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range. The results indicate a B-like conformation for each of the strands in the stem part of the PS hairpin and reverse Watson-Crick base pairing between the T and A residues. These data are consistent with a previously calculated structure for parallel-stranded DNA

New theories for smectic and nematic liquid-crystal polymers: Backbone LCPs [liquid crystalline polymers] and their mixtures and side-chain LCPs

International Nuclear Information System (INIS)

Dowell, F.

1987-01-01

A summary of predictions and explanations from statistical-physics theories for both backbone and side-chain liquid crystalline polymers (LCPs) and for mixtures with backbone LCPs are presented. Trends in the thermodynamic and molecular ordering properties have been calculated as a function of pressure, density, temperature, and molecule chemical structures (including degree of polymerization and the following properties of the chemical structures of the repeat units: lengths and shapes, intra-chain rotation energies, dipole moments, site-site polarizabilities and Lennard-Jones potentials, etc.) in nematic and multiple smectic-A LC phases and in the isotropic liquid phase. The theoretical results are found to be in good agreement with existing experimental data. These theories can also be applied to combined LCPs. Since these theories have no ad hoc or arbitrarily adjustable parameters, these theories can be used to design new LCPs and new solvents as well as to predict and explain properties. 27 refs., 4 tabs

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow.

Science.gov (United States)

Castelain, Mickaël; Duviau, Marie-Pierre; Oxaran, Virginie; Schmitz, Philippe; Cocaign-Bousquet, Muriel; Loubière, Pascal; Piard, Jean-Christophe; Mercier-Bonin, Muriel

2016-09-01

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.

Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

Directory of Open Access Journals (Sweden)

Yunpeng Zhang

2015-01-01

Full Text Available Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it is important to identify subtype specific miRNA-mRNA modules. In this study, we integrated the Ping-Pong algorithm and multiobjective genetic algorithm to identify subtype specific miRNA-mRNA functional regulatory modules (MFRMs through integrative analysis of three biological data sets: GO biological processes, miRNA target information, and matched miRNA and mRNA expression data. We applied our method on a heterogeneous disease, multiple myeloma (MM, to identify MM subtype specific MFRMs. The constructed miRNA-mRNA regulatory networks provide modular outlook at subtype specific miRNA-mRNA interactions. Furthermore, clustering analysis demonstrated that heterogeneous MFRMs were able to separate corresponding MM subtypes. These subtype specific MFRMs may aid in the further elucidation of the pathogenesis of each subtype and may serve to guide MM subtype diagnosis and treatment.

8-Oxo-7,8-dihydroadenine and 8-Oxo-7,8-dihydroadenosine-Chemistry, Structure, and Function in RNA and Their Presence in Natural Products and Potential Drug Derivatives.

Science.gov (United States)

Choi, Yu Jung; Chang, Stephanie J; Gibala, Krzysztof S; Resendiz, Marino J E

2017-05-17

A description and history of the role that 8-oxo-7,8-dihydroadenine (8-oxoAde) and 8-oxo-7,8-dihydroadenosine (8-oxoA) have in various fields has been compiled. This Review focusses on 1) the formation of this oxidatively generated modification in RNA, its interactions with other biopolymers, and its potential role in the development/progression of disease; 2) the independent synthesis and incorporation of this modified nucleoside into oligonucleotides of RNA to display the progress that has been made in establishing its behavior in biologically relevant systems; 3) reported synthetic routes, which date back to 1890, along with the progress that has been made in the total synthesis of the nucleobase, nucleoside, and their corresponding derivatives; and 4) the isolation, total synthesis, and biological activity of natural products containing these moieties as the backbone. The current state of research regarding this oxidatively generated lesion as well as its importance in the context of RNA, natural products, and potential as drug derivatives is illustrated using all available examples reported to date. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlation between protein secondary structure, backbone bond angles, and side-chain orientations

Science.gov (United States)

Lundgren, Martin; Niemi, Antti J.

2012-08-01

We investigate the fine structure of the sp3 hybridized covalent bond geometry that governs the tetrahedral architecture around the central Cα carbon of a protein backbone, and for this we develop new visualization techniques to analyze high-resolution x-ray structures in the Protein Data Bank. We observe that there is a correlation between the deformations of the ideal tetrahedral symmetry and the local secondary structure of the protein. We propose a universal coarse-grained energy function to describe the ensuing side-chain geometry in terms of the Cβ carbon orientations. The energy function can model the side-chain geometry with a subatomic precision. As an example we construct the Cα-Cβ structure of HP35 chicken villin headpiece. We obtain a configuration that deviates less than 0.4 Å in root-mean-square distance from the experimental x-ray structure.

Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

Science.gov (United States)

Kis, András; Tholt, Gergely; Ivanics, Milán; Várallyay, Éva; Jenes, Barnabás; Havelda, Zoltán

2016-04-01

Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect-mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature. In this study, we designed artificial microRNAs (amiRNAs) using a barley miRNA precursor backbone, which target different conservative sequence elements of the WDV strains. Potential amiRNA sequences were selected to minimize the off-target effects and were tested in a transient sensor system in order to select the most effective constructs at low temperature. On the basis of the data obtained, a polycistronic amiRNA precursor construct (VirusBuster171) was built expressing three amiRNAs simultaneously. The construct was transformed into barley under the control of a constitutive promoter. The transgenic lines were kept at 12-15 °C to mimic autumn and spring conditions in which major WDV infection and accumulation take place. We were able to establish a stable barley transgenic line displaying resistance to insect-mediated WDV infection. Our study demonstrates that amiRNA technology can be an efficient tool for the introduction of highly efficient resistance in barley against a DNA virus belonging to the Geminiviridae family, and this resistance is effective at low temperature where the natural insect vector mediates the infection process. © 2015 BSPP and John Wiley & Sons Ltd.

antaRNA: ant colony-based RNA sequence design.

Science.gov (United States)

Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

2015-10-01

RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found ,: inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology ,: reliable RNA sequence design becomes a crucial step to generate novel biochemical components. In this article ,: the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution ,: specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. http://www.bioinf.uni-freiburg.de/Software/antaRNA CONTACT: backofen@informatik.uni-freiburg.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

Science.gov (United States)

Michelini, Flavia; Jalihal, Ameya P; Francia, Sofia; Meers, Chance; Neeb, Zachary T; Rossiello, Francesca; Gioia, Ubaldo; Aguado, Julio; Jones-Weinert, Corey; Luke, Brian; Biamonti, Giuseppe; Nowacki, Mariusz; Storici, Francesca; Carninci, Piero; Walter, Nils G; Fagagna, Fabrizio d'Adda di

2018-03-30

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

Data acquisition backbone core DABC release v1.0

Energy Technology Data Exchange (ETDEWEB)

Adamczewski-Musch, Joern; Essel, Hans G.; Kurz, Nikolaus; Linev, S. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany)

2010-07-01

The new experiments at FAIR require new concepts of data acquisition systems for the distribution of self-triggered, time stamped data streams over high performance networks for event building. The Data Acquisition Backbone Core (DABC) is a general purpose software framework developed for the implementation of such data acquisition systems. A DABC application consists of functional components like data input, combiner, scheduler, event builder, filter, analysis and storage which can be configured at runtime. Application specific code including the support of all kinds of data channels (front-end systems) is implemented by C++ program plug-ins. DABC is also well suited as environment for various detector and readout components test beds. A set of DABC plug-ins has been developed for the FAIR experiment CBM (Compressed Baryonic Matter) at GSI. This DABC application is used as DAQ system for test beamtimes. Front-end boards equipped with n-XYTER ASICs and ADCs are connected to read-out controller boards (ROC). From there the data is sent over Ethernet (UDP), or over optics and PCIe interface cards into Linux PCs. DABC does the controlling, event building, archiving and data serving. The first release of DABC was published in 2009 and is available under GPL license.

34A, miRNA-944, miRNA-101 and miRNA-218 in cervical cancer

African Journals Online (AJOL)

RNAs (21 - 24 nucleotides in length) that are critical for many important processes such as development, ... RNA extraction and reverse transcription. Total RNA was extracted from each of the experimental groups using ... used as an endogenous control to normalize the expression of miRNA-143, miRNA-34A, miRNA-.

Protein backbone motions viewed by intraresidue and sequential H{sup N}-H{sup {alpha}} residual dipolar couplings

Energy Technology Data Exchange (ETDEWEB)

Voegeli, Beat; Yao Lishan; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: bax@nih.gov

2008-05-15

Triple resonance E.COSY-based techniques were used to measure intra-residue and sequential H{sup N}-H{sup {alpha}} residual dipolar couplings (RDCs) for the third IgG-binding domain of protein G (GB3), aligned in Pf1 medium. Measurements closely correlate with values predicted on the basis of an NMR structure, previously determined on the basis of a large number of one-bond backbone RDCs measured in five alignment media. However, in particular the sequential H{sup N}-H{sup {alpha}} RDCs are smaller than predicted for a static structure, suggesting a degree of motion for these internuclear vectors that exceeds that of the backbone amide N-H vectors. Of all experimentally determined GB3 structures available, the best correlation between experimental {sup 1}H-{sup 1}H couplings is observed for a GB3 ensemble, previously derived to generate a realistic picture of the conformational space sampled by GB3 (Clore and Schwieters, J Mol Biol 355:879-886, 2006). However, for both NMR and X-ray-derived structures the {sup 1}H-{sup 1}H couplings are found to be systematically smaller than expected on the basis of alignment tensors derived from {sup 15}N-{sup 1}H amide RDCs, assuming librationally corrected N-H bond lengths of 1.041 A.

Solid state radiation chemistry of the DNA backbone

International Nuclear Information System (INIS)

Bernhard, W.A.

1989-09-01

The long term goal of this program is to determine the fundamental rules needed to predict the type and yield of damage produced in DNA due to direct effects of ionizing radiation. The focus is on damage to the sugar-phosphate backbone, damage that would lead to strand breaks. Model systems have been chosen that permit various aspects of this problem to be investigated. The emphasis will be on single crystals of monosaccharides, nucleosides, and nucleotides but will also include some powder work on polynucleotides. In these model systems, free radical products and reactions are observed by electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) techniques. The information thus gained is used in constructing rules that predict what primary free radicals are formed in single crystals of model compounds and the reactions stemming from the primary radicals. The formulation of a set of rules that work in model systems will represent a major advance toward formulating a set of rules that predict the direct damage in DNA itself. In a broader context this program is part of the effort to understand and predict the effects of exposure to ionizing radiation received at low dose rates over long periods of time. Assessment of low dose effects requires a basic understanding of the action of radiation at the molecular level. By contributing to that basic understanding, this program will help solve the problems of risk assessment under low dose conditions. 5 refs., 3 figs

Extracellular RNA Communication (ExRNA)

Data.gov (United States)

Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

RNA Crystallization

Science.gov (United States)

Golden, Barbara L.; Kundrot, Craig E.

2003-01-01

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

Identifying microRNA/mRNA dysregulations in ovarian cancer.

Science.gov (United States)

Miles, Gregory D; Seiler, Michael; Rodriguez, Lorna; Rajagopal, Gunaretnam; Bhanot, Gyan

2012-03-27

MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Our findings identify

Natural RNA circles function as efficient microRNA sponges

DEFF Research Database (Denmark)

Hansen, Thomas Birkballe; Jensen, Trine I; Clausen, Bettina Hjelm

2013-01-01

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called comp......MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so......-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more...... sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA....

RNA Origami

DEFF Research Database (Denmark)

Sparvath, Steffen Lynge

introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

Branched RNA: A New Architecture for RNA Interference

Directory of Open Access Journals (Sweden)

Anna Aviñó

2011-01-01

Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

Heterogeneous self-assembled media for biopolymerization

DEFF Research Database (Denmark)

Monnard, Pierre-Alain

2011-01-01

Heterogeneous media, such as micro-structured aqueous environments, could offer an alternative approach to the synthesis of biopolymers with novel functions. Structured media are here defined as specialized, self-assembled structures that are formed, e.g, by amphiphiles, such as liposomes, emulsion...... polymerization, the initial elongation rates clearly depended on the complementarity of the monomers with the templating nucleobases3. However, metal-ion catalyzed reactions deliver RNA analogs with heterogeneous linkages. Moreover, the usefulness of this medium in the form of quasi-compartmentalization extends...... beyond metal-ion catalysis reactions, as we have recently demonstrated the catalytic power of a dipeptide, SerHis, for the regioselective formation of phosphodiester bonds. These results in conjonction with the synthesis of nucleobases at -78˚C, the demonstration of ribozyme activity (RNA ligase ribozyme...

Plant RNA Regulatory Network and RNA Granules in Virus Infection

Directory of Open Access Journals (Sweden)

Kristiina Mäkinen

2017-12-01

Full Text Available Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

Science.gov (United States)

Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

2017-01-01

Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual

Methylated nucleosides in tRNA and tRNA methyltransferases

Directory of Open Access Journals (Sweden)

Hiroyuki eHori

2014-05-01

Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

Polystyrene Backbone Polymers Consisting of Alkyl-Substituted Triazine Side Groups for Phosphorescent OLEDs

Directory of Open Access Journals (Sweden)

Beatrice Ch. D. Salert

2012-01-01

Full Text Available This paper describes the synthesis of new electron-transporting styrene monomers and their corresponding polystyrenes all with a 2,4,6-triphenyl-1,3,5-triazine basic structure in the side group. The monomers differ in the alkyl substitution and in the meta-/paralinkage of the triazine to the polymer backbone. The thermal and spectroscopic properties of the new electron-transporting polymers are discussed in regard to their chemical structures. Phosphorescent OLEDs were prepared using the obtained electron-transporting polymers as the emissive layer material in blend systems together with a green iridium-based emitter 13 and a small molecule as an additional cohost with wideband gap characteristics (CoH-001. The performance of the OLEDs was characterized and discussed in regard to the chemical structure of the new electron-transporting polymers.

RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

International Nuclear Information System (INIS)

Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E.; Eghbalnia, Hamid R.

2012-01-01

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ( 1 H– 15 N 2D HMQC) and proton–proton nuclear Overhauser enhancement spectroscopy ( 1 H– 1 H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a

RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

Energy Technology Data Exchange (ETDEWEB)

Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E. [National Magnetic Resonance Facility at Madison (United States); Eghbalnia, Hamid R., E-mail: eghbalhd@uc.edu [University of Cincinnati, Department of Molecular and Cellular Physiology (United States)

2012-04-15

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ({sup 1}H-{sup 15}N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ({sup 1}H-{sup 1}H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino

Extensive Air Showers: from the muonic smoking guns to the hadronic backbone

Directory of Open Access Journals (Sweden)

Cazon L.

2013-06-01

Full Text Available Extensive Air Showers are complex macroscopic objects initiated by single ultra-high energy particles. They are the result of millions of high energy reactions in the atmosphere and can be described as the superposition of hadronic and electromagnetic cascades. The hadronic cascade is the air shower backbone, and it is mainly made of pions. Decays of neutral pions initiate electromagnetic cascades, while the decays of charged pions produce muons which leave the hadronic core and travel many kilometers almost unaffected. Muons are smoking guns of the hadronic cascade: the energy, transverse momentum, spatial distribution and depth of production are key to reconstruct the history of the air shower. In this work, we overview the phenomenology of muons on the air shower and its relation to the hadronic cascade. We briefly review the experimental efforts to analyze muons within air showers and discuss possible paths to use this information.

MysiRNA-designer: a workflow for efficient siRNA design.

Directory of Open Access Journals (Sweden)

Mohamed Mysara

Full Text Available The design of small interfering RNA (siRNA is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.

Backbone of complex networks of corporations: The flow of control

Science.gov (United States)

Glattfelder, J. B.; Battiston, S.

2009-09-01

We present a methodology to extract the backbone of complex networks based on the weight and direction of links, as well as on nontopological properties of nodes. We show how the methodology can be applied in general to networks in which mass or energy is flowing along the links. In particular, the procedure enables us to address important questions in economics, namely, how control and wealth are structured and concentrated across national markets. We report on the first cross-country investigation of ownership networks, focusing on the stock markets of 48 countries around the world. On the one hand, our analysis confirms results expected on the basis of the literature on corporate control, namely, that in Anglo-Saxon countries control tends to be dispersed among numerous shareholders. On the other hand, it also reveals that in the same countries, control is found to be highly concentrated at the global level, namely, lying in the hands of very few important shareholders. Interestingly, the exact opposite is observed for European countries. These results have previously not been reported as they are not observable without the kind of network analysis developed here.

RNA binding and replication by the poliovirus RNA polymerase

International Nuclear Information System (INIS)

Oberste, M.S.

1988-01-01

RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

RNA.

Science.gov (United States)

Darnell, James E., Jr.

1985-01-01

Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

OpenAIRE

Zhang, Yunpeng; Liu, Wei; Xu, Yanjun; Li, Chunquan; Wang, Yingying; Yang, Haixiu; Zhang, Chunlong; Su, Fei; Li, Yixue; Li, Xia

2015-01-01

Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it i...

microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

Science.gov (United States)

Pinder, Benjamin D; Smibert, Craig A

2013-01-01

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

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Andronescu Mirela

2008-08-01

Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

MicroRNA from tuberculosis RNA: A bioinformatics study

OpenAIRE

Wiwanitkit, Somsri; Wiwanitkit, Viroj

2012-01-01

The role of microRNA in the pathogenesis of pulmonary tuberculosis is the interesting topic in chest medicine at present. Recently, it was proposed that the microRNA can be a useful biomarker for monitoring of pulmonary tuberculosis and might be the important part in pathogenesis of disease. Here, the authors perform a bioinformatics study to assess the microRNA within known tuberculosis RNA. The microRNA part can be detected and this can be important key information in further study of the p...

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

Science.gov (United States)

Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

Studying RNA-protein interactions in vivo by RNA immunoprecipitation

DEFF Research Database (Denmark)

Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

2011-01-01

and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

RNA SURVEILLANCE– AN EMERGING ROLE FOR RNA REGULATORY NETWORKS IN AGING

OpenAIRE

Montano, Monty; Long, Kimberly

2010-01-01

In this review, we describe recent advances in the field of RNA regulatory biology and relate these advances to aging science. We introduce a new term, RNA surveillance, an RNA regulatory process that is conserved in metazoans, and describe how RNA surveillance represents molecular cross-talk between two emerging RNA regulatory systems – RNA interference and RNA editing. We discuss how RNA surveillance mechanisms influence mRNA and microRNA expression and activity during lifespan. Additionall...

Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

NARCIS (Netherlands)

Kluiver, Joost; Gibcus, Johan H.; Hettinga, Chris; Adema, Annelies; Richter, Mareike K. S.; Halsema, Nancy; Slezak-Prochazka, Izabella; Ding, Ye; Kroesen, Bart-Jan; van den Berg, Anke

2012-01-01

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and

High affinity RNA targeting by oligonucleotides displaying aromatic stacking and amino groups in the major groove. Comparison of triazoles and phenylsubstituents

DEFF Research Database (Denmark)

Kumar, Pawan; Hornum, Mick; Nielsen, Lise Junker

2014-01-01

Three 5-modified 2'-deoxyuridine nucleosides were synthesized and incorporated into oligonucleotides and compared with the previously published 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine monomer W. The introduction of an aminomethyl group on the phenyl group led to monomer X, which was found...... to thermally stabilize a 9-mer DNA:RNA duplex, presumably through the partial neutralization of the negative charge of the backbone. By also taking advantage of the stacking interactions in the major groove of two or more of the monomer X, an extremely high thermal stability was obtained. A regioisomer...... monomer Z was incorporated for comparison, and it was found to give a more neutral influence on duplex stability indicating less efficient stacking interactions. The duplexes were investigated by CD spectroscopy and MD simulations....

Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

Science.gov (United States)

Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

2008-08-22

NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

Science.gov (United States)

Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

2010-04-08

RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

Plant RNA binding proteins for control of RNA virus infection

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Sung Un eHuh

2013-12-01

Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

APSY-NMR for protein backbone assignment in high-throughput structural biology

Energy Technology Data Exchange (ETDEWEB)

Dutta, Samit Kumar; Serrano, Pedro; Proudfoot, Andrew; Geralt, Michael [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States); Pedrini, Bill [Paul Scherrer Institute (PSI), SwissFEL Project (Switzerland); Herrmann, Torsten [Université de Lyon, Institut des Sciences Analytiques, Centre de RMN à Très Hauts Champs, UMR 5280 CNRS, ENS Lyon, UCB Lyon 1 (France); Wüthrich, Kurt, E-mail: wuthrich@scripps.edu [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States)

2015-01-15

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [{sup 1}H,{sup 1}H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.

Effective Anti-miRNA Oligonucleotides Show High Releasing Rate of MicroRNA from RNA-Induced Silencing Complex.

Science.gov (United States)

Ariyoshi, Jumpei; Matsuyama, Yohei; Kobori, Akio; Murakami, Akira; Sugiyama, Hiroshi; Yamayoshi, Asako

2017-10-01

MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonucleotides (AMOs), which are composed of the 2'-O-methyl (2'-OMe) RNA, could induce the release of miRNA from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs, which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the relationship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC 50 ) using conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are directly proportional to the IC 50 values, and AMOs, which have an ability to promote the release of miRNA from RISC, can effectively inhibit RISC activity in living cells.

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

DEFF Research Database (Denmark)

Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

2007-01-01

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

Predicting backbone Cα angles and dihedrals from protein sequences by stacked sparse auto-encoder deep neural network.

Science.gov (United States)

Lyons, James; Dehzangi, Abdollah; Heffernan, Rhys; Sharma, Alok; Paliwal, Kuldip; Sattar, Abdul; Zhou, Yaoqi; Yang, Yuedong

2014-10-30

Because a nearly constant distance between two neighbouring Cα atoms, local backbone structure of proteins can be represented accurately by the angle between C(αi-1)-C(αi)-C(αi+1) (θ) and a dihedral angle rotated about the C(αi)-C(αi+1) bond (τ). θ and τ angles, as the representative of structural properties of three to four amino-acid residues, offer a description of backbone conformations that is complementary to φ and ψ angles (single residue) and secondary structures (>3 residues). Here, we report the first machine-learning technique for sequence-based prediction of θ and τ angles. Predicted angles based on an independent test have a mean absolute error of 9° for θ and 34° for τ with a distribution on the θ-τ plane close to that of native values. The average root-mean-square distance of 10-residue fragment structures constructed from predicted θ and τ angles is only 1.9Å from their corresponding native structures. Predicted θ and τ angles are expected to be complementary to predicted ϕ and ψ angles and secondary structures for using in model validation and template-based as well as template-free structure prediction. The deep neural network learning technique is available as an on-line server called Structural Property prediction with Integrated DEep neuRal network (SPIDER) at http://sparks-lab.org. Copyright © 2014 Wiley Periodicals, Inc.

IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions.

Science.gov (United States)

Mann, Martin; Wright, Patrick R; Backofen, Rolf

2017-07-03

The IntaRNA algorithm enables fast and accurate prediction of RNA-RNA hybrids by incorporating seed constraints and interaction site accessibility. Here, we introduce IntaRNAv2, which enables enhanced parameterization as well as fully customizable control over the prediction modes and output formats. Based on up to date benchmark data, the enhanced predictive quality is shown and further improvements due to more restrictive seed constraints are highlighted. The extended web interface provides visualizations of the new minimal energy profiles for RNA-RNA interactions. These allow a detailed investigation of interaction alternatives and can reveal potential interaction site multiplicity. IntaRNAv2 is freely available (source and binary), and distributed via the conda package manager. Furthermore, it has been included into the Galaxy workflow framework and its already established web interface enables ad hoc usage. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

Science.gov (United States)

Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

2018-05-01

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

International Nuclear Information System (INIS)

Bodkin, D.K.

1985-01-01

RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

Directory of Open Access Journals (Sweden)

Baodong Wu

2009-03-01

Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

AUTOBA: automation of backbone assignment from HN(C)N suite of experiments.

Science.gov (United States)

Borkar, Aditi; Kumar, Dinesh; Hosur, Ramakrishna V

2011-07-01

Development of efficient strategies and automation represent important milestones of progress in rapid structure determination efforts in proteomics research. In this context, we present here an efficient algorithm named as AUTOBA (Automatic Backbone Assignment) designed to automate the assignment protocol based on HN(C)N suite of experiments. Depending upon the spectral dispersion, the user can record 2D or 3D versions of the experiments for assignment. The algorithm uses as inputs: (i) protein primary sequence and (ii) peak-lists from user defined HN(C)N suite of experiments. In the end, one gets H(N), (15)N, C(α) and C' assignments (in common BMRB format) for the individual residues along the polypeptide chain. The success of the algorithm has been demonstrated, not only with experimental spectra recorded on two small globular proteins: ubiquitin (76 aa) and M-crystallin (85 aa), but also with simulated spectra of 27 other proteins using assignment data from the BMRB.

Bifurcations in the interplay of messenger RNA, protein and nonprotein coding RNA

International Nuclear Information System (INIS)

Zhdanov, Vladimir P

2008-01-01

The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-protein association resulting in suppression of the protein regulatory activity or (ii) ncRNA-mRNA association resulting in degradation of the miRNA-mRNA complex. The kinetic models describing these two scenarios are found to predict bistability provided that protein suppresses the ncRNA formation

A density functional study of backbone structures of polydiacetylene: destabilization of butatriene structure

International Nuclear Information System (INIS)

Katagiri, Hideki; Shimoi, Yukihiro; Abe, Shuji

2004-01-01

Backbone structures of polydiacetylene are studied with first-principles electronic structure method using plane-waves within generalized gradient approximation (GGA) of density functional theory. In spin-restricted calculations a coarse k-point sampling gives a potential energy curve with two local minima corresponding to acetylene and butatriene structures. However, the potential barrier between the two structures rapidly decreases with increasing number of k-points, which results in destabilization of the butatriene structure. Spin polarization effects also destabilize the butatriene structure, inducing atom-centered spin-density-wave state. These potential energies were compared with those obtained by Hartree-Fock, density functional within local density approximation (LDA) and GGA, and hybrid density functional methods using a gaussian basis set. The comparison shows that the density functional methods within LDA and GGA favor the destabilization of the butatriene structure in contrast to the Hartree-Fock method

Experimental Tracking of Limit-Point Bifurcations and Backbone Curves Using Control-Based Continuation

Science.gov (United States)

Renson, Ludovic; Barton, David A. W.; Neild, Simon A.

Control-based continuation (CBC) is a means of applying numerical continuation directly to a physical experiment for bifurcation analysis without the use of a mathematical model. CBC enables the detection and tracking of bifurcations directly, without the need for a post-processing stage as is often the case for more traditional experimental approaches. In this paper, we use CBC to directly locate limit-point bifurcations of a periodically forced oscillator and track them as forcing parameters are varied. Backbone curves, which capture the overall frequency-amplitude dependence of the system’s forced response, are also traced out directly. The proposed method is demonstrated on a single-degree-of-freedom mechanical system with a nonlinear stiffness characteristic. Results are presented for two configurations of the nonlinearity — one where it exhibits a hardening stiffness characteristic and one where it exhibits softening-hardening.

How the RNA isolation method can affect microRNA microarray results

DEFF Research Database (Denmark)

Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

2011-01-01

RNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform micro...

The integrated analysis of RNA-seq and microRNA-seq depicts miRNA-mRNA networks involved in Japanese flounder (Paralichthys olivaceus) albinism.

Science.gov (United States)

Wang, Na; Wang, Ruoqing; Wang, Renkai; Tian, Yongsheng; Shao, Changwei; Jia, Xiaodong; Chen, Songlin

2017-01-01

Albinism, a phenomenon characterized by pigmentation deficiency on the ocular side of Japanese flounder (Paralichthys olivaceus), has caused significant damage. Limited mRNA and microRNA (miRNA) information is available on fish pigmentation deficiency. In this study, a high-throughput sequencing strategy was employed to identify the mRNA and miRNAs involved in P. olivaceus albinism. Based on P. olivaceus genome, RNA-seq identified 21,787 know genes and 711 new genes by transcripts assembly. Of those, 235 genes exhibited significantly different expression pattern (fold change ≥2 or ≤0.5 and q-value≤0.05), including 194 down-regulated genes and 41 up-regulated genes in albino versus normally pigmented individuals. These genes were enriched to 81 GO terms and 9 KEGG pathways (p≤0.05). Among those, the pigmentation related pathways-Melanogenesis and tyrosine metabolism were contained. High-throughput miRNA sequencing identified a total of 475 miRNAs, including 64 novel miRNAs. Furthermore, 33 differentially expressed miRNAs containing 13 up-regulated and 20 down-regulated miRNAs were identified in albino versus normally pigmented individuals (fold change ≥1.5 or ≤0.67 and p≤0.05). The next target prediction discovered a variety of putative target genes, of which, 134 genes including Tyrosinase (TYR), Tyrosinase-related protein 1 (TYRP1), Microphthalmia-associated transcription factor (MITF) were overlapped with differentially expressed genes derived from RNA-seq. These target genes were significantly enriched to 254 GO terms and 103 KEGG pathways (p<0.001). Of those, tyrosine metabolism, lysosomes, phototransduction pathways, etc., attracted considerable attention due to their involvement in regulating skin pigmentation. Expression patterns of differentially expressed mRNA and miRNAs were validated in 10 mRNA and 10 miRNAs by qRT-PCR. With high-throughput mRNA and miRNA sequencing and analysis, a series of interested mRNA and miRNAs involved in fish

Switching off small RNA regulation with trap-mRNA

DEFF Research Database (Denmark)

Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

2009-01-01

to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

Science.gov (United States)

Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

2010-11-01

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

Science.gov (United States)

Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

2014-09-25

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Cytoplasmic Z-RNA

International Nuclear Information System (INIS)

Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

1987-01-01

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

SOX2 and OCT4 mRNA-expressing cells, detected by molecular beacons, localize to the center of neurospheres during differentiation.

Directory of Open Access Journals (Sweden)

Mirolyuba Ilieva

Full Text Available Neurospheres are used as in vitro assay to measure the properties of neural stem cells. To investigate the molecular and phenotypic heterogeneity of neurospheres, molecular beacons (MBs targeted against the stem cell markers OCT4 and SOX2 were designed, and synthesized with a 2'-O-methyl RNA backbone. OCT4 and SOX2 MBs were transfected into human embryonic mesencephalon derived cells, which spontaneously form neurospheres when grown on poly-L-ornitine/fibronectin matrix and medium complemented with bFGF. OCT4 and SOX2 gene expression were tracked in individual cell using the MBs. Quantitative image analysis every day for seven days showed that the OCT4 and SOX2 mRNA-expressing cells clustered in the centre of the neurospheres cultured in differentiation medium. By contrast, cells at the periphery of the differentiating spheres developed neurite outgrowths and expressed the tyrosine hydroxylase protein, indicating terminal differentiation. Neurospheres cultured in growth medium contained OCT4 and SOX2-positive cells distributed throughout the entire sphere, and no differentiating neurones. Gene expression of SOX2 and OCT4 mRNA detected by MBs correlated well with gene and protein expression measured by qRT-PCR and immunostaining, respectively. These experimental data support the theoretical model that stem cells cluster in the centre of neurospheres, and demonstrate the use of MBs for the spatial localization of specific gene-expressing cells within heterogeneous cell populations.

RNA Interference - Towards RNA becoming a Medicine -42 ...

Indian Academy of Sciences (India)

research. A brief history of the development ofRNAi is shown in. Box 2. Mechanism of ... new RNA strand using target RNA as the template and thereby converting it ... thought to excise precursor stRNA from their -70 nt stem loop precursor to ...

Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

International Nuclear Information System (INIS)

Hamilton, R.W.; Lloyd, R.S.

1989-01-01

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or sliding mechanism on non-target DNA as opposed to a distributive or random hit mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0

Competition Between Co(NH3)63+ and Inner Sphere Mg2+ Ions in the HDV Ribozyme

Science.gov (United States)

Gong, Bo; Chen, Jui-Hui; Bevilacqua, Philip C.; Golden, Barbara L.; Carey, Paul R.

2009-01-01

Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg2+ are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH3)63+, an analog of Mg(H2O)62+. Here, the binding of Mg2+ and Co(NH3)63+ to the HDV ribozyme are studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg2+ alone is introduced to the ribozyme, inner sphere coordination of Mg(H2O)x2+ (x≤5) to non-bridging PO2− oxygen, and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg2+ induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH3)63+ alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg2+ binding, Co(NH3)63+ binding does not perturb PO2− group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH3)63+ ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO2− groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH3)63+ ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg2+-PO2− binding sites: sites that Co(NH3)63+ can displace, and others it cannot. PMID:19888753

Role of RNase MRP in viral RNA degradation and RNA recombination.

Science.gov (United States)

Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

2011-01-01

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

Assignment of protein backbone resonances using connectivity, torsion angles and 13Cα chemical shifts

International Nuclear Information System (INIS)

Morris, Laura C.; Valafar, Homayoun; Prestegard, James H.

2004-01-01

A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just 13 C α chemical shifts (δ( 13 C α )) and data restricting the φ and ψ backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in δ( 13 C α ), φ, and ψ space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and 13 C α chemical shift data are available

Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

Energy Technology Data Exchange (ETDEWEB)

Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

1996-05-01

Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition ¹⁵N T₁, T₂, and ¹⁵N/¹H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone ¹H, ¹³C, and ¹⁵N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and ¹⁵N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Köster, Tino

2017-04-13

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-01-01

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

Bovine oocytes and early embryos express mRNA encoding glycerol kinase but addition of glycerol to the culture media interferes with oocyte maturation.

Science.gov (United States)

Okawara, Sumika; Hamano, Seizo; Tetsuka, Masafumi

2009-04-01

Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.

RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

NARCIS (Netherlands)

Dorssers, L.

1983-01-01

The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

Previously, an RNA-dependent RNA polymerase produced upon infection of

An Unusual Conformational Isomer of Verrucosidin Backbone from a Hydrothermal Vent Fungus, Penicillium sp. Y-50-10.

Science.gov (United States)

Pan, Chengqian; Shi, Yutong; Auckloo, Bibi Nazia; Chen, Xuegang; Chen, Chen-Tung Arthur; Tao, Xinyi; Wu, Bin

2016-08-18

A new verrucosidin derivative, methyl isoverrucosidinol (1), was isolated from the marine fungus Penicillium sp. Y-50-10, dwelling in sulfur rich sediment in the Kueishantao hydrothermal vents off Taiwan. The structure was established by spectroscopic means including HRMS and 2D-NMR spectroscopic analysis. The absolute configuration was defined mainly by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Among hitherto known compounds with a verrucosidine backbone isolated from natural resource, compound 1 represents the first example of a new conformational isomer of its skeleton, exhibiting antibiotic activity against Bacillus subtilis with MIC value 32 μg/mL.

Rigidity of the polypeptide backbone in the triple-stranded collagen molecule.

Science.gov (United States)

Nemethy, G

1981-02-01

Conformational energy computations were carried out on collagen-like triple-stranded conformations of several polytripeptides with the structure CH3CO(GXY)3NHCH3, where X and Y can be Pro, Ala, or Gly. The computed minimum-energy conformations for various sequences are compared with that computed earlier for poly(Gly-Pro-Pro). Usually, substitution of Ala or Gly residues for Pro does not cause any strain or distortion of the conformation of the triple-stranded complex. Thus, the structure is a very stable and essentially rigid one. Unfavorable interactions were found only in the case of CH3CO(Gly-Ala-Pro)NHCH3. These interactions are a consequence of differences between the residue geometry of Ala and Pro. They result in small changes of some backbone dihedral angles and in an increase of intra- and interchain energies. The presence of a single Gly-Ala-Pro tripeptide within a sequence of Gly-Pro-Pro tripeptides is not sufficient, however, to cause even a small distoration of the triple strand. No deviation of the peptide groups from planarity is required to stabilize the triple-stranded structure.

Processing of UV-induced DNA damage in diverse biological systems

International Nuclear Information System (INIS)

Galloway, A.M.

1992-01-01

A novel protocol has been developed allowing direct evaluation and accurate quantitation of UV lesions contained with both genomic DNA and the small oligonucleotides excised by a living cell during nucleotide excision repair. Using this methodology, the repair capacity of normal and UV-sensitive cells of human, Chinese hamster, and Escherichia coli origin, has been assessed. Several conclusions have been reached: (1) severage of the interpyrimidine phosphodiester linkage of cyclobutane dimers appears to be an evolutionarily conserved phenomenon; (2) the kinetics of cyclobutane dimer repair differ markedly from both (6-4) photoproduct and TA* lesion removal; (3) the ability to excise cyclobutane dimers is independent of (6-4) photoproduct repair capacity, suggesting that the lesions are not repaired/recognized by identical mechanisms; (4) fibroblast strains representing the eight xeroderma pigmentosum complementation groups each show a unique proficiency/deficiency to repair the different photolesions under study, implicating that a defect in a different locus underlies each genetic form of this disease; (5) the repair deficiency in UV-sensitive strains of trichothiodystrophy appears to be completely unrelated to that of non-complementing XP-D cells. To allow direct assessment of an IDP-altered photoproduct, substrates have been constructed which contain, at a defined dithymidine site, no lesion, a conventional cyclobutane dimer, or a cyclobutane dimer modified by severage of the intradimer phosphodiester bond. Bacteriophage T4 UV endonuclease has no activity towards a modified lesion, questioning the interpretation of experiments which utilize the strand-incising activity of this enzyme to monitor repair. Furthermore, although this altered lesion acts as a block to E. coli DNA polymerase I, it allows SP6 RNA polymerase to bypass the otherwise RNA polymerase-blocking lesion

Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase.

Science.gov (United States)

Heck, Julie A; Meng, Xiao; Frick, David N

2009-04-01

Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111-22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

Fast prediction of RNA-RNA interaction using heuristic algorithm.

Science.gov (United States)

Montaseri, Soheila

2015-01-01

Interaction between two RNA molecules plays a crucial role in many medical and biological processes such as gene expression regulation. In this process, an RNA molecule prohibits the translation of another RNA molecule by establishing stable interactions with it. Some algorithms have been formed to predict the structure of the RNA-RNA interaction. High computational time is a common challenge in most of the presented algorithms. In this context, a heuristic method is introduced to accurately predict the interaction between two RNAs based on minimum free energy (MFE). This algorithm uses a few dot matrices for finding the secondary structure of each RNA and binding sites between two RNAs. Furthermore, a parallel version of this method is presented. We describe the algorithm's concurrency and parallelism for a multicore chip. The proposed algorithm has been performed on some datasets including CopA-CopT, R1inv-R2inv, Tar-Tar*, DIS-DIS, and IncRNA54-RepZ in Escherichia coli bacteria. The method has high validity and efficiency, and it is run in low computational time in comparison to other approaches.

The RNA synthesis machinery of negative-stranded RNA viruses

International Nuclear Information System (INIS)

Ortín, Juan; Martín-Benito, Jaime

2015-01-01

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

The RNA synthesis machinery of negative-stranded RNA viruses

Energy Technology Data Exchange (ETDEWEB)

Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

2015-05-15

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

Infrared and swelling study of wet-spun films of DNA\\cdot[(bipy)Pt(en)]^2+ as a function of hydration

Science.gov (United States)

Kuebler, A.; Whitson, K. B.; Marlowe, R. L.; Lee, S. A.; Rupprecht, A.

1997-11-01

A ladder-like conformation of DNA has been induced by the binding of bipyridyl-Pt(II)-ethylenediamine (denoted as [(bipy)Pt(en)]^2+) at a relative humidity (RH) of 75%.(Arnott et al.,) Nature 287, (1980) 561. We report the first study of oriented films of this complex as a function of hydration by using Fourier transform infrared (IR) spectroscopy and optical microscopy. Vibrational modes between 800 and 1000 cm-1 are localized in the phosphodiester backbone and are sensitive to changes in the geometry of the molecule. The IR spectrum of this region is substantially different at 59% RH than at higher humidities, implying that a conformational change occurs as the RH is lowered below 75%. Optical microscopy measurements of small pieces of films of the complex also show changes at low RH. These trends are consistent with an order-disorder transition occurring as the RH is lowered The measurements also show that the DNA\\cdot[(bipy)Pt(en)]^2+) are very stable at the highest humidities.

Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

KAUST Repository

Tsutakawa, Susan E.

2017-06-27

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5\\'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5\\'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5\\'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering\\', basic residues energetically steer an inverted ss 5\\'-flap through a gateway over FEN1\\'s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5\\'-flap specificity and catalysis, preventing genomic instability.

Synthesis and properties of ApA analogues with shortened phosphonate internucleotide linkage.

Science.gov (United States)

Králíková, Sárka; Buděšínský, Miloš; Barvík, Ivan; Masojídková, Milena; Točík, Zdeněk; Rosenberg, Ivan

2011-01-01

A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH(2)-C4 ″ linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.

Clinical significance of LUNX mRNA, CK19 mRNA, CEA mRNA expression in detecting micrometastasis from lung cancer

International Nuclear Information System (INIS)

Zhu Guangying; Liu Delin; Chen Jie

2003-01-01

Objective: To evaluate the sensitivity, specificity and clinical significance of CK19 mRNA, CEA mRNA and LUNX mRNA for detecting micrometastasis by sampling the peripheral blood and regional lymph nodes of lung cancer patients. Methods: Reverse transcriptase chain reaction (RT-PCR) was used to detect LUNX mRNA, CK19 mRNA, CEA mRNA for micrometastasis by sampling the peripheral blood of 48 lung cancer patients and 44 regional lymph nodes of such patients treated by curative resection. Peripheral blood of 30 patients with pulmonary benign lesions and 10 normal healthy volunteers and lymph nodes of 6 patients with benign pulmonary diseases served as control. Results: 1) LUNX mRNA, CK19 mRNA, CEA mRNA were expressed in all (35/35) lung cancer tissues. 2) In the peripheral blood from 48 lung cancer patients, 30 (62.5%) were positive for LUNX mRNA, 24 (50.0%) positive for CK19 mRNA and 32(66.7%) positive for CEA mRNA. The positive detection rates of micrometastasis in 44 lymph nodes from lung cancer patients were 36.4% (16 out of 44) for LUNX mRNA, 27.3% (12 out of 44) for CK19 mRNA and 40.9% (18 out of 44) for CEA mRNA. 3) In the 30 blood samples from patients with pulmonary benign diseases, 2 (6.7%) expressed CK19 mRNA, but none expressed LUNX mRNA or CEA mRNA. All the 3 molecular markers were negative in the 10 blood samples from healthy volunteers. In 11 lymph nodes from patients with pulmonary benign lesions, none was positive for any of the three markers. 4) In 44 regional lymph nodes from lung cancer patients, 6 (13.6%) were positive for metastasis by histopathological examination, with a positive rate significantly lower than that of the RT-PCR (P<0.05). 5) The micrometastatic positive rate in the peripheral blood of 40 non-small cell lung cancer (NSCLC) patients was significantly related to TNM stage (P=0.01). Conclusions: LUNX mRNA, CK19 MRNA, CEA mRNA are all appropriate target genes for the detection of micrometastasis from lung cancer. LUNX mRNA and CEA mRNA

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

Science.gov (United States)

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

Directory of Open Access Journals (Sweden)

Joanna J Moser

2010-10-01

Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

Cancer-Related Triplets of mRNA-lncRNA-miRNA Revealed by Integrative Network in Uterine Corpus Endometrial Carcinoma

Directory of Open Access Journals (Sweden)

Chenglin Liu

2017-01-01

Full Text Available The regulation of transcriptome expression level is a complex process involving multiple-level interactions among molecules such as protein coding RNA (mRNA, long noncoding RNA (lncRNA, and microRNA (miRNA, which are essential for the transcriptome stability and maintenance and regulation of body homeostasis. The availability of multilevel expression data enables a comprehensive view of the regulatory network. In this study, we analyzed the coding and noncoding gene expression profiles of 301 patients with uterine corpus endometrial carcinoma (UCEC. A new method was proposed to construct a genome-wide integrative network based on variance inflation factor (VIF regression method. The cross-regulation relations of mRNA, lncRNA, and miRNA were then selected based on clique-searching algorithm from the network, when any two molecules of the three were shown as interacting according to the integrative network. Such relation, which we call the mRNA-lncRNA-miRNA triplet, demonstrated the complexity in transcriptome regulation process. Finally, six UCEC-related triplets were selected in which the mRNA participates in endometrial carcinoma pathway, such as CDH1 and TP53. The multi-type RNAs are proved to be cross-regulated as to each of the six triplets according to literature. All the triplets demonstrated the association with the initiation and progression of UCEC. Our method provides a comprehensive strategy for the investigation of transcriptome regulation mechanism.

Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

International Nuclear Information System (INIS)

Kolakofsky, Daniel; Le Mercier, Philippe; Iseni, Frederic; Garcin, Dominique

2004-01-01

mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in that the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template

The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans

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Manuel Michaël

2010-02-01

Full Text Available Abstract Background Comparative genomics of the early diverging metazoan lineages and of their unicellular sister-groups opens new window to reconstructing the genetic changes which preceded or accompanied the evolution of multicellular body plans. A recent analysis found that the genome of the nerve-less sponges encodes the homologues of most vertebrate post-synaptic proteins. In vertebrate excitatory synapses, these proteins assemble to form the post-synaptic density, a complex molecular platform linking membrane receptors, components of their signalling pathways, and the cytoskeleton. Newly available genomes from Monosiga brevicollis (a member of Choanoflagellata, the closest unicellular relatives of animals and Trichoplax adhaerens (a member of Placozoa: besides sponges, the only nerve-less metazoans offer an opportunity to refine our understanding of post-synaptic protein evolution. Results Searches for orthologous proteins and reconstruction of gene gains/losses based on the taxon phylogeny indicate that post-synaptic proteins originated in two main steps. The backbone scaffold proteins (Shank, Homer, DLG and some of their partners were acquired in a unicellular ancestor of choanoflagellates and metazoans. A substantial additional set appeared in an exclusive ancestor of the Metazoa. The placozoan genome contains most post-synaptic genes but lacks some of them. Notably, the master-scaffold protein Shank might have been lost secondarily in the placozoan lineage. Conclusions The time of origination of most post-synaptic proteins was not concomitant with the acquisition of synapses or neural-like cells. The backbone of the scaffold emerged in a unicellular context and was probably not involved in cell-cell communication. Based on the reconstructed protein composition and potential interactions, its ancestral function could have been to link calcium signalling and cytoskeleton regulation. The complex later became integrated into the evolving

On RNA-RNA interaction structures of fixed topological genus.

Science.gov (United States)

Fu, Benjamin M M; Han, Hillary S W; Reidys, Christian M

2015-04-01

Interacting RNA complexes are studied via bicellular maps using a filtration via their topological genus. Our main result is a new bijection for RNA-RNA interaction structures and a linear time uniform sampling algorithm for RNA complexes of fixed topological genus. The bijection allows to either reduce the topological genus of a bicellular map directly, or to lose connectivity by decomposing the complex into a pair of single stranded RNA structures. Our main result is proved bijectively. It provides an explicit algorithm of how to rewire the corresponding complexes and an unambiguous decomposition grammar. Using the concept of genus induction, we construct bicellular maps of fixed topological genus g uniformly in linear time. We present various statistics on these topological RNA complexes and compare our findings with biological complexes. Furthermore we show how to construct loop-energy based complexes using our decomposition grammar. Copyright © 2015 Elsevier Inc. All rights reserved.

An Enhanced Backbone-Assisted Reliable Framework for Wireless Sensor Networks

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Amna Ali

2010-03-01

Full Text Available An extremely reliable source to sink communication is required for most of the contemporary WSN applications especially pertaining to military, healthcare and disaster-recovery. However, due to their intrinsic energy, bandwidth and computational constraints, Wireless Sensor Networks (WSNs encounter several challenges in reliable source to sink communication. In this paper, we present a novel reliable topology that uses reliable hotlines between sensor gateways to boost the reliability of end-to-end transmissions. This reliable and efficient routing alternative reduces the number of average hops from source to the sink. We prove, with the help of analytical evaluation, that communication using hotlines is considerably more reliable than traditional WSN routing. We use reliability theory to analyze the cost and benefit of adding gateway nodes to a backbone-assisted WSN. However, in hotline assisted routing some scenarios where source and the sink are just a couple of hops away might bring more latency, therefore, we present a Signature Based Routing (SBR scheme. SBR enables the gateways to make intelligent routing decisions, based upon the derived signature, hence providing lesser end-to-end delay between source to the sink communication. Finally, we evaluate our proposed hotline based topology with the help of a simulation tool and show that the proposed topology provides manifold increase in end-to-end reliability.

Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

DEFF Research Database (Denmark)

Hansen, M.C.; Nielsen, A.K.; Molin, Søren

2001-01-01

obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes...

Thermoresponsive Poly(2-oxazoline) Molecular Brushes by Living Ionic Polymerization: Kinetic Investigations of Pendant Chain Grafting and Cloud Point Modulation by Backbone and Side Chain Length Variation

KAUST Repository

Zhang, Ning; Luxenhofer, Robert; Jordan, Rainer

2012-01-01

and the stretched conformation of the backbone, which is caused by the electrostatic repulsion of the oxazolinium moieties along the macroinitiator. The resulting molecular brushes showed thermoresponsive properties, that is, having a defined cloud point (CP

RNA-Catalyzed Polymerization and Replication of RNA

Science.gov (United States)

Horning, D. P.; Samantha, B.; Tjhung, K. F.; Joyce, G. F.

2017-07-01

In an effort to reconstruct RNA-based life, in vitro evolution was used to obtain an RNA polymerase ribozyme that can synthesize a variety of complex functional RNAs and can catalyze the exponential amplification of short RNAs.

Defibrotide, a polydisperse mixture of single-stranded phosphodiester oligonucleotides with lifesaving activity in severe hepatic veno-occlusive disease: clinical outcomes and potential mechanisms of action.

Science.gov (United States)

Kornblum, Noah; Ayyanar, Kanyalakshmi; Benimetskaya, Luba; Richardson, Paul; Iacobelli, Massimo; Stein, C A

2006-01-01

Veno-occlusive disease of the liver (VOD) remains a troubling and potentially fatal complication of high-dose chemotherapy and hematopoietic stem cell transplantation conditioning regimens. No effective therapy has been available for these patients to date, and the best supportive care measures remain woefully inadequate. Defibrotide (DF) (Gentium, S.p.A., Como, Italy), a polydisperse mixture of all the single-stranded phosphodiester oligodeoxyribonucleotides that can be obtained from the controlled depolymerization of porcine intestinal mucosal genomic DNA, seems to offer a safe and effective treatment for some patients suffering from severe VOD, a condition for which no accepted standard therapy currently exists. Early clinical studies evaluating the efficacy of DF for the treatment of severe VOD in patients undergoing hematopoietic stem cell transplantation have been very encouraging. Approximately 45% of the patients treated in multiple initial phase II clinical trials achieved a complete response at day +100, demonstrating normalization of serum bilirubin and resolution of the clinical syndrome. However, although multi-institutional, these represented single arm studies. A large, FDA-approved, pivotal, prospective, multi-institutional, global phase III trial of DF vs. historical controls (best available therapy) commenced in the first quarter of 2006 and should provide further validation of DF's efficacy. The drug seems to have few significant side effects, and almost all test subjects who have received this treatment have tolerated it well. Although the mechanism of action remains unclear, the drug exerts minimal systemic anticoagulant effects yet appears to induce numerous antithrombotic and profibrinolytic effects both in vitro and in vivo. It may function as an adenosine receptor agonist and causes increased concentrations of endogenous prostaglandins, which modulate thrombomodulin, platelets, and fibrinolysis. It also appears to block lipopolysaccharide

microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

OpenAIRE

Pinder, Benjamin D; Smibert, Craig A

2012-01-01

Argonaute 1 directly interacts with the RNA binding protein Smaug in Drosophila, is thereby recruited to the Smaug target nanos mRNA and is required for Smaug-mediated translational repression of the nanos mRNA.

TargetRNA: a tool for predicting targets of small RNA action in bacteria

OpenAIRE

Tjaden, Brian

2008-01-01

Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-...

The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

Science.gov (United States)

Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

2011-02-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeting MicroRNAs with Small Molecules a Novel Approach to Treating Breast Cancer

Science.gov (United States)

2011-10-01

or deoxyribozyme, is a catalytic DNA that site-specifically cleaves the target RNA Watson – Crick base pairing to a complementary target sequence...RNA A-helix fold among the selected pre- miRNA targets. Furthermore, 3D characteristics including Watson - Crick base pairs and wobble base pairs...phosphorothioate backbone in addition to 2′-O-methoxyethyl AMOs are ASOs against miRNAs and therefore produce ASO–miRNA duplexes through Watson – Crick binding

RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

Science.gov (United States)

Haque, Farzin; Pi, Fengmei; Zhao, Zhengyi; Gu, Shanqing; Hu, Haibo; Yu, Hang; Guo, Peixuan

2018-01-01

In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs. © 2017 Wiley Periodicals, Inc.

MicroRNA-target binding structures mimic microRNA duplex structures in humans.

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Xi Chen

Full Text Available Traditionally, researchers match a microRNA guide strand to mRNA sequences using sequence comparisons to predict its potential target genes. However, many of the predictions can be false positives due to limitations in sequence comparison alone. In this work, we consider the association of two related RNA structures that share a common guide strand: the microRNA duplex and the microRNA-target binding structure. We have analyzed thousands of such structure pairs and found many of them share high structural similarity. Therefore, we conclude that when predicting microRNA target genes, considering just the microRNA guide strand matches to gene sequences may not be sufficient--the microRNA duplex structure formed by the guide strand and its companion passenger strand must also be considered. We have developed software to translate RNA binding structure into encoded representations, and we have also created novel automatic comparison methods utilizing such encoded representations to determine RNA structure similarity. Our software and methods can be utilized in the other RNA secondary structure comparisons as well.

TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

Science.gov (United States)

Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole

International Nuclear Information System (INIS)

Krizova, Hana; Zidek, Lukas; Stone, Martin J.; Novotny, Milos V.; Sklenar, Vladimir

2004-01-01

Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by 15 N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2-sec-4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature depencence of the motional parameters

Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

DEFF Research Database (Denmark)

Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

2015-01-01

) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

An Unusual Conformational Isomer of Verrucosidin Backbone from a Hydrothermal Vent Fungus, Penicillium sp. Y-50-10

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Chengqian Pan

2016-08-01

Full Text Available A new verrucosidin derivative, methyl isoverrucosidinol (1, was isolated from the marine fungus Penicillium sp. Y-50-10, dwelling in sulfur rich sediment in the Kueishantao hydrothermal vents off Taiwan. The structure was established by spectroscopic means including HRMS and 2D-NMR spectroscopic analysis. The absolute configuration was defined mainly by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Among hitherto known compounds with a verrucosidine backbone isolated from natural resource, compound 1 represents the first example of a new conformational isomer of its skeleton, exhibiting antibiotic activity against Bacillus subtilis with MIC value 32 μg/mL.

A Method to Predict the Structure and Stability of RNA/RNA Complexes.

Science.gov (United States)

Xu, Xiaojun; Chen, Shi-Jie

2016-01-01

RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Intermolecular loop-loop base pairing is a widespread and functionally important tertiary structure motif in RNA machinery. However, computational prediction of intermolecular loop-loop base pairing is challenged by the entropy and free energy calculation due to the conformational constraint and the intermolecular interactions. In this chapter, we describe a recently developed statistical mechanics-based method for the prediction of RNA/RNA complex structures and stabilities. The method is based on the virtual bond RNA folding model (Vfold). The main emphasis in the method is placed on the evaluation of the entropy and free energy for the loops, especially tertiary kissing loops. The method also uses recursive partition function calculations and two-step screening algorithm for large, complicated structures of RNA/RNA complexes. As case studies, we use the HIV-1 Mal dimer and the siRNA/HIV-1 mutant (T4) to illustrate the method.

Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

Science.gov (United States)

Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

2007-04-01

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection

International Nuclear Information System (INIS)

Kleinschmidt, A.M.; Pederson, T.

1990-01-01

The authors present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32 P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells

RNA precursor pool metabolism and RNA synthesis in X-irradiated Tetrahymena

International Nuclear Information System (INIS)

Stephens, R.E.; Paul, I.J.; Zimmerman, A.M.

1976-01-01

The incorporation of a radioactive RNA precursor ( 3 H-uridine) has been used in many studies as an index for measuring the synthesis of RNA, yet there is a distinct possibility that the results so obtained were significantly influenced by radiation-induced effects on the metabolism of this precursor into UTP (the primary immediate precursor of RNA) before its incorporation into RNA. A direct examination was therefore undertaken of the effects of X-irradiation on the metabolism of 3 H-uridine and its relationship to RNA synthesis as determined by incorporation. X-irradiation of logarithmically growing Tetrahymena pyriformis caused a dose-dependent depression of total cellular RNA synthesis. Ribosomal RNA (which comprises about 80 per cent of total cellular RNA) synthesis was also depressed by X-irradiation in a dose-dependent manner. Measurements of the levels of radioactivity present in the UTP precursor pool of both irradiated and unirradiated cells were obtained by means of DEAE-cellulose column chromatography of the extracted free nucleotides. Metabolism of 3 H-uridine into UMP, UDP and UTP was depressed by 40%, 26% and 27% respectively, whereas incorporation of 3 H-uridine into RNA was depressed by 77%. The results show that about one-third of the observed (apparent) depression in RNA synthesis was due to radiation-induced effects on the precursor pool, and the remaining two-thirds due to some definite effect of radiation at the transcription level leading to depressed synthesis of RNA. (U.K.)

Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury.

Directory of Open Access Journals (Sweden)

David M Rissin

Full Text Available We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA probe-containing a reactive amine instead of a nucleotide at a specific position in the sequence-for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase-containing an aldehyde group-that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG, the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122-an established biomarker of liver toxicity-extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.

Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

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Nicole Ludwig

2016-03-01

Full Text Available Wilms tumor (WT is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

Energy Technology Data Exchange (ETDEWEB)

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D. (Scripps)

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

dPORE-miRNA: Polymorphic regulation of microRNA genes

KAUST Repository

Schmeier, Sebastian; Schaefer, Ulf; MacPherson, Cameron R.; Bajic, Vladimir B.

2011-01-01

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

dPORE-miRNA: Polymorphic regulation of microRNA genes

KAUST Repository

Schmeier, Sebastian

2011-02-04

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

Chemical synthesis of membrane proteins by the removable backbone modification method.

Science.gov (United States)

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

International Nuclear Information System (INIS)

Armstrong, R.D.; Cadman, E.C.

1985-01-01

Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

RNA decay by messenger RNA interferases

DEFF Research Database (Denmark)

Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

2008-01-01

Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

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Alexey Berezhnoy

2012-01-01

Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

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Song Cai

2011-07-01

Full Text Available Abstract Background Siraitia grosvenorii (Luohanguo is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. Results In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9% unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450 and ninety UDP-glucosyltransferase (UDPG unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. Conclusion A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

Science.gov (United States)

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

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Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

2010-01-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

Science.gov (United States)

Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

2014-01-01

Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

RegRNA: an integrated web server for identifying regulatory RNA motifs and elements

OpenAIRE

Huang, Hsi-Yuan; Chien, Chia-Hung; Jen, Kuan-Hua; Huang, Hsien-Da

2006-01-01

Numerous regulatory structural motifs have been identified as playing essential roles in transcriptional and post-transcriptional regulation of gene expression. RegRNA is an integrated web server for identifying the homologs of regulatory RNA motifs and elements against an input mRNA sequence. Both sequence homologs and structural homologs of regulatory RNA motifs can be recognized. The regulatory RNA motifs supported in RegRNA are categorized into several classes: (i) motifs in mRNA 5′-untra...

Large-scale phylogenomic analysis resolves a backbone phylogeny in ferns

Science.gov (United States)

Shen, Hui; Jin, Dongmei; Shu, Jiang-Ping; Zhou, Xi-Le; Lei, Ming; Wei, Ran; Shang, Hui; Wei, Hong-Jin; Zhang, Rui; Liu, Li; Gu, Yu-Feng; Zhang, Xian-Chun; Yan, Yue-Hong

2018-01-01

Abstract Background Ferns, originated about 360 million years ago, are the sister group of seed plants. Despite the remarkable progress in our understanding of fern phylogeny, with conflicting molecular evidence and different morphological interpretations, relationships among major fern lineages remain controversial. Results With the aim to obtain a robust fern phylogeny, we carried out a large-scale phylogenomic analysis using high-quality transcriptome sequencing data, which covered 69 fern species from 38 families and 11 orders. Both coalescent-based and concatenation-based methods were applied to both nucleotide and amino acid sequences in species tree estimation. The resulting topologies are largely congruent with each other, except for the placement of Angiopteris fokiensis, Cheiropleuria bicuspis, Diplaziopsis brunoniana, Matteuccia struthiopteris, Elaphoglossum mcclurei, and Tectaria subpedata. Conclusions Our result confirmed that Equisetales is sister to the rest of ferns, and Dennstaedtiaceae is sister to eupolypods. Moreover, our result strongly supported some relationships different from the current view of fern phylogeny, including that Marattiaceae may be sister to the monophyletic clade of Psilotaceae and Ophioglossaceae; that Gleicheniaceae and Hymenophyllaceae form a monophyletic clade sister to Dipteridaceae; and that Aspleniaceae is sister to the rest of the groups in eupolypods II. These results were interpreted with morphological traits, especially sporangia characters, and a new evolutionary route of sporangial annulus in ferns was suggested. This backbone phylogeny in ferns sets a foundation for further studies in biology and evolution in ferns, and therefore in plants. PMID:29186447

Large-scale phylogenomic analysis resolves a backbone phylogeny in ferns.

Science.gov (United States)

Shen, Hui; Jin, Dongmei; Shu, Jiang-Ping; Zhou, Xi-Le; Lei, Ming; Wei, Ran; Shang, Hui; Wei, Hong-Jin; Zhang, Rui; Liu, Li; Gu, Yu-Feng; Zhang, Xian-Chun; Yan, Yue-Hong

2018-02-01

Ferns, originated about 360 million years ago, are the sister group of seed plants. Despite the remarkable progress in our understanding of fern phylogeny, with conflicting molecular evidence and different morphological interpretations, relationships among major fern lineages remain controversial. With the aim to obtain a robust fern phylogeny, we carried out a large-scale phylogenomic analysis using high-quality transcriptome sequencing data, which covered 69 fern species from 38 families and 11 orders. Both coalescent-based and concatenation-based methods were applied to both nucleotide and amino acid sequences in species tree estimation. The resulting topologies are largely congruent with each other, except for the placement of Angiopteris fokiensis, Cheiropleuria bicuspis, Diplaziopsis brunoniana, Matteuccia struthiopteris, Elaphoglossum mcclurei, and Tectaria subpedata. Our result confirmed that Equisetales is sister to the rest of ferns, and Dennstaedtiaceae is sister to eupolypods. Moreover, our result strongly supported some relationships different from the current view of fern phylogeny, including that Marattiaceae may be sister to the monophyletic clade of Psilotaceae and Ophioglossaceae; that Gleicheniaceae and Hymenophyllaceae form a monophyletic clade sister to Dipteridaceae; and that Aspleniaceae is sister to the rest of the groups in eupolypods II. These results were interpreted with morphological traits, especially sporangia characters, and a new evolutionary route of sporangial annulus in ferns was suggested. This backbone phylogeny in ferns sets a foundation for further studies in biology and evolution in ferns, and therefore in plants. © The Authors 2017. Published by Oxford University Press.

Strategies underlying RNA silencing suppression by negative strand RNA viruses

NARCIS (Netherlands)

Hemmes, J.C.

2007-01-01

The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double

Selecting Question-Specific Genes to Reduce Incongruence in Phylogenomics: A Case Study of Jawed Vertebrate Backbone Phylogeny.

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Chen, Meng-Yun; Liang, Dan; Zhang, Peng

2015-11-01

Incongruence between different phylogenomic analyses is the main challenge faced by phylogeneticists in the genomic era. To reduce incongruence, phylogenomic studies normally adopt some data filtering approaches, such as reducing missing data or using slowly evolving genes, to improve the signal quality of data. Here, we assembled a phylogenomic data set of 58 jawed vertebrate taxa and 4682 genes to investigate the backbone phylogeny of jawed vertebrates under both concatenation and coalescent-based frameworks. To evaluate the efficiency of extracting phylogenetic signals among different data filtering methods, we chose six highly intractable internodes within the backbone phylogeny of jawed vertebrates as our test questions. We found that our phylogenomic data set exhibits substantial conflicting signal among genes for these questions. Our analyses showed that non-specific data sets that are generated without bias toward specific questions are not sufficient to produce consistent results when there are several difficult nodes within a phylogeny. Moreover, phylogenetic accuracy based on non-specific data is considerably influenced by the size of data and the choice of tree inference methods. To address such incongruences, we selected genes that resolve a given internode but not the entire phylogeny. Notably, not only can this strategy yield correct relationships for the question, but it also reduces inconsistency associated with data sizes and inference methods. Our study highlights the importance of gene selection in phylogenomic analyses, suggesting that simply using a large amount of data cannot guarantee correct results. Constructing question-specific data sets may be more powerful for resolving problematic nodes. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

Science.gov (United States)

Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

2010-11-01

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

Science.gov (United States)

Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

2018-03-19

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

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Darrin V. Bann

2012-06-01

Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA.

Science.gov (United States)

Parker, Greg S; Eckert, Debra M; Bass, Brenda L

2006-05-01

In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.

Reconstruction and analysis of the lncRNA-miRNA-mRNA network based on competitive endogenous RNA reveal functional lncRNAs in rheumatoid arthritis.

Science.gov (United States)

Jiang, Hui; Ma, Rong; Zou, Shubiao; Wang, Yongzhong; Li, Zhuqing; Li, Weiping

2017-06-01

Rheumatoid arthritis (RA) is an autoimmune disease with an unknown etiology, occurring in approximately 1.0% of general population. More and more studies have suggested that long non-coding RNAs (lncRNAs) could play important roles in various biological processes and be associated with the pathogenesis of different kinds of diseases including RA. Although a large number of lncRNAs have been found, our knowledge of their function and physiological/pathological significance is still in its infancy. In order to reveal functional lncRNAs and identify the key lncRNAs in RA, we reconstructed a global triple network based on the competitive endogenous RNA (ceRNA) theory using the data from National Center for Biotechnology Information Gene Expression Omnibus and our previous paper. Meanwhile, Gene Ontology (GO) and pathway analysis were performed using Cytoscape plug-in BinGO and Database for Annotation, Visualization, and Integration Discovery (DAVID), respectively. We found that the lncRNA-miRNA-mRNA network was composed of 7 lncRNA nodes, 90 mRNA nodes, 24 miRNA nodes, and 301 edges. The functional assay showed that 147 GO terms and 23 pathways were enriched. In addition, three lncRNAs (S5645.1, XR_006437.1, J01878) were highly related to RA, and therefore, were selected as key lncRNAs. This study suggests that specific lncRNAs are associated with the development of RA, and three lncRNAs (S5645.1, XR_006437.1, J01878) could be used as potential diagnostic biomarkers and therapeutic targets.

Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication.

Science.gov (United States)

Mizumoto, Hiroyuki; Iwakawa, Hiro-Oki; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

2006-04-01

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

Influence of the strength of the smectic order on the backbone anisotropy of side-chain liquid crystal polymers as revealed by SANS

Science.gov (United States)

Noirez, L.; Keller, P.; Cotton, J. P.

1992-06-01

It is proposed that the strength of the smectic order determines the backbone anisotropy of side-chain liquid crystal polymers. Here this strength increases with the length of the alkyl terminal group of the mesogens. Two liquid crystal polymethacrylates differing only by the mesogenic tails —OCH3 and —OC4H9 are considered. The backbone anisotropy of these polymers is measured by small angle neutron scattering (SANS) whereas the smectic order is evaluated from the intensity of the 001 Bragg peak. Il est proposé que la qualité de l'ordre smectique détermine l'anisotropie du squelette de polymères mésomorphes en peigne confinés dans les lamelles. Ici l'ordre smectique est augmenté en allongeant le groupe alkyl terminal des mésogènes. Nous étudions deux polyméthacrylates cristal liquide qui ne différent que par leurs groupes terminaux : —OCH3 et —OC4H9. L'anisotropie du squellete est mesurée par diffusion de neutrons aux petits angles tandis que l'ordre smectique est évalué à l'aide de l'intensité du pic de Bragg 001.

Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

Directory of Open Access Journals (Sweden)

Matteo A Avella

Full Text Available Endogenous microbiota play essential roles in the host's immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP, higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group. We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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Brittney R Henderson

Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles.

Science.gov (United States)

Haque, Farzin; Guo, Peixuan

2015-01-01

RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149-155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833-842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applications in nanomedicine and nanotechnology. Rapid advances in RNA chemistry, RNA biophysics, and RNA biology have created new opportunities for translating basic science into clinical practice. RNA nanotechnology holds considerable promise in this regard. Increased evidence also suggests that substantial part of the 98.5 % of human genome (Lander et al. Nature 409:860-921, 2001) that used to be called "junk DNA" actually codes for noncoding RNA. As we understand more on how RNA structures are related to function, we can fabricate synthetic RNA nanoparticles for the diagnosis and treatment of diseases. This chapter provides a brief overview of the field regarding the design, construction, purification, and characterization of RNA nanoparticles for diverse applications in nanotechnology and nanomedicince.

The ViennaRNA web services.

Science.gov (United States)

Gruber, Andreas R; Bernhart, Stephan H; Lorenz, Ronny

2015-01-01

The ViennaRNA package is a widely used collection of programs for thermodynamic RNA secondary structure prediction. Over the years, many additional tools have been developed building on the core programs of the package to also address issues related to noncoding RNA detection, RNA folding kinetics, or efficient sequence design considering RNA-RNA hybridizations. The ViennaRNA web services provide easy and user-friendly web access to these tools. This chapter describes how to use this online platform to perform tasks such as prediction of minimum free energy structures, prediction of RNA-RNA hybrids, or noncoding RNA detection. The ViennaRNA web services can be used free of charge and can be accessed via http://rna.tbi.univie.ac.at.

RNA-SeQC: RNA-seq metrics for quality control and process optimization.

Science.gov (United States)

DeLuca, David S; Levin, Joshua Z; Sivachenko, Andrey; Fennell, Timothy; Nazaire, Marc-Danie; Williams, Chris; Reich, Michael; Winckler, Wendy; Getz, Gad

2012-06-01

RNA-seq, the application of next-generation sequencing to RNA, provides transcriptome-wide characterization of cellular activity. Assessment of sequencing performance and library quality is critical to the interpretation of RNA-seq data, yet few tools exist to address this issue. We introduce RNA-SeQC, a program which provides key measures of data quality. These metrics include yield, alignment and duplication rates; GC bias, rRNA content, regions of alignment (exon, intron and intragenic), continuity of coverage, 3'/5' bias and count of detectable transcripts, among others. The software provides multi-sample evaluation of library construction protocols, input materials and other experimental parameters. The modularity of the software enables pipeline integration and the routine monitoring of key measures of data quality such as the number of alignable reads, duplication rates and rRNA contamination. RNA-SeQC allows investigators to make informed decisions about sample inclusion in downstream analysis. In summary, RNA-SeQC provides quality control measures critical to experiment design, process optimization and downstream computational analysis. See www.genepattern.org to run online, or www.broadinstitute.org/rna-seqc/ for a command line tool.

Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine

Directory of Open Access Journals (Sweden)

Chiranjib Chakraborty

2017-09-01

Full Text Available In the past few years, therapeutic microRNA (miRNA and small interfering RNA (siRNA are some of the most important biopharmaceuticals that are in commercial space as future medicines. This review summarizes the patents of miRNA- and siRNA-based new drugs, and also provides a snapshot about significant biopharmaceutical companies that are investing for the therapeutic development of miRNA and siRNA molecules. An insightful view about individual siRNA and miRNA drugs has been depicted with their present status, which is gaining attention in the therapeutic landscape. The efforts of the biopharmaceuticals are discussed with the status of their preclinical and/or clinical trials. Here, some of the setbacks have been highlighted during the biopharmaceutical development of miRNA and siRNA as individual therapeutics. Finally, a snapshot is illustrated about pharmacokinetics, pharmacodynamics with absorption, distribution, metabolism, and excretion (ADME, which is the fundamental development process of these therapeutics, as well as the delivery system for miRNA- and siRNA-based drugs. Keywords: miRNA, siRNA, drug development

Resolution of deep nodes yields an improved backbone phylogeny and a new basal lineage to study early evolution of Asteraceae.

Science.gov (United States)

Panero, Jose L; Freire, Susana E; Ariza Espinar, Luis; Crozier, Bonnie S; Barboza, Gloria E; Cantero, Juan J

2014-11-01

A backbone phylogeny that fully resolves all subfamily and deeper nodes of Asteraceae was constructed using 14 chloroplast DNA loci. The recently named genus Famatinanthus was found to be sister to the Mutisioideae-Asteroideae clade that represents more than 99% of Asteraceae and was found to have the two chloroplast inversions present in all Asteraceae except the nine genera of Barnadesioideae. A monotypic subfamily Famatinanthoideae and tribe Famatinantheae are named herein as new. Relationships among the basal lineages of the family were resolved with strong support in the Bayesian analysis as (Barnadesioideae (Famatinanthoideae (Mutisioideae (Stifftioideae (Wunderlichioideae-Asteroideae))))). Ancestral state reconstruction of ten morphological characters at the root node of the Asteraceae showed that the ancestral sunflower would have had a woody habit, alternate leaves, solitary capitulescences, epaleate receptacles, smooth styles, smooth to microechinate pollen surface sculpturing, white to yellow corollas, and insect-mediated pollination. Herbaceous habit, echinate pollen surface, pubescent styles, and cymose capitulescences were reconstructed for backbone nodes of the phylogeny corresponding to clades that evolved shortly after Asteraceae dispersed out of South America. No support was found for discoid capitula, multiseriate involucres or bird pollination as the ancestral character condition for any node. Using this more resolved phylogenetic tree, the recently described Raiguenrayun cura+Mutisiapollis telleriae fossil should be associated to a more derived node than previously suggested when time calibrating phylogenies of Asteraceae. Copyright © 2014 Elsevier Inc. All rights reserved.

5S rRNA-derived and tRNA-derived SINEs in fruit bats.

Science.gov (United States)

Gogolevsky, Konstantin P; Vassetzky, Nikita S; Kramerov, Dmitri A

2009-05-01

Most short retroposons (SINEs) descend from cellular tRNA of 7SL RNA. Here, four new SINEs were found in megabats (Megachiroptera) but neither in microbats nor in other mammals. Two of them, MEG-RS and MEG-RL, descend from another cellular RNA, 5S rRNA; one (MEG-T2) is a tRNA-derived SINE; and MEG-TR is a hybrid tRNA/5S rRNA SINE. Insertion locus analysis suggests that these SINEs were active in the recent fruit bat evolution. Analysis of MEG-RS and MEG-RL in comparison with other few 5S rRNA-derived SINEs demonstrates that the internal RNA polymerase III promoter is their most invariant region, while the secondary structure is more variable. The mechanisms underlying the modular structure of these and other SINEs as well as their variation are discussed. The scenario of evolution of MEG SINEs is proposed.

Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

Science.gov (United States)

de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

2016-01-01

Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

Science.gov (United States)

Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

2014-02-04

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

Science.gov (United States)

Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

2015-05-06

RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

The early history of tRNA recognition by aminoacyl-tRNA synthetases

Indian Academy of Sciences (India)

Madhu

2006-10-04

Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

Science.gov (United States)

Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

2014-05-01

RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

Science.gov (United States)

Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

2018-03-05

Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

A path-based measurement for human miRNA functional similarities using miRNA-disease associations

Science.gov (United States)

Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao

2016-09-01

Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

Coupling between myosin head conformation and the thick filament backbone structure.

Science.gov (United States)

Hu, Zhongjun; Taylor, Dianne W; Edwards, Robert J; Taylor, Kenneth A

2017-12-01

The recent high-resolution structure of the thick filament from Lethocerus asynchronous flight muscle shows aspects of thick filament structure never before revealed that may shed some light on how striated muscles function. The phenomenon of stretch activation underlies the function of asynchronous flight muscle. It is most highly developed in flight muscle, but is also observed in other striated muscles such as cardiac muscle. Although stretch activation is likely to be complex, involving more than a single structural aspect of striated muscle, the thick filament itself, would be a prime site for regulatory function because it must bear all of the tension produced by both its associated myosin motors and any externally applied force. Here we show the first structural evidence that the arrangement of myosin heads within the interacting heads motif is coupled to the structure of the thick filament backbone. We find that a change in helical angle of 0.16° disorders the blocked head preferentially within the Lethocerus interacting heads motif. This observation suggests a mechanism for how tension affects the dynamics of the myosin heads leading to a detailed hypothesis for stretch activation and shortening deactivation, in which the blocked head preferentially binds the thin filament followed by the free head when force production occurs. Copyright © 2017 Elsevier Inc. All rights reserved.

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

Science.gov (United States)

Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

2015-01-29

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

RNase-assisted RNA chromatography

Science.gov (United States)

Michlewski, Gracjan; Cáceres, Javier F.

2010-01-01

RNA chromatography combined with mass spectrometry represents a widely used experimental approach to identify RNA-binding proteins that recognize specific RNA targets. An important drawback of most of these protocols is the high background due to direct or indirect nonspecific binding of cellular proteins to the beads. In many cases this can hamper the detection of individual proteins due to their low levels and/or comigration with contaminating proteins. Increasing the salt concentration during washing steps can reduce background, but at the cost of using less physiological salt concentrations and the likely loss of important RNA-binding proteins that are less stringently bound to a given RNA, as well as the disassembly of protein or ribonucleoprotein complexes. Here, we describe an improved RNA chromatography method that relies on the use of a cocktail of RNases in the elution step. This results in the release of proteins specifically associated with the RNA ligand and almost complete elimination of background noise, allowing a more sensitive and thorough detection of RNA-binding proteins recognizing a specific RNA transcript. PMID:20571124

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

Directory of Open Access Journals (Sweden)

Kim Chan-Mi

2007-07-01

Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

RNA modifications by oxidation

DEFF Research Database (Denmark)

Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

2012-01-01

to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

Science.gov (United States)

Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

2016-01-01

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

Staggering in the cleavage pattern of E. coli ABC-excinuclease

International Nuclear Information System (INIS)

Myles, G.M.; Van Houten, B.; Sancar, A.

1986-01-01

E. coli ABC excinuclease is a complex of three proteins encoded by the uvrA, uvrB, and uvrC genes. The enzyme repairs DNA mono and diadducts by the single strand cleavage of DNA eight phosphodiester bond 5' and four or five phosphodiester bonds 3' to a DNA lesion and facilitates the removal of the resulting twelve or thirteen nucleotide fragment. In this study, the authors have investigated the excision pattern for ultraviolet (UV) induced diadducts, i.e. cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts. Terminally (5' or 3') labeled DNA was irradiated with 254nm UV and treated with ABC excinuclease before and after photoreactivation of cyclobutane dimers by E. coli DNA photolyase. In this way, the authors were able to differentiate between the cleavage pattern of pyrimidine dimers and of (6-4) photoproducts. Their results show that certain TT cyclobutane dimers and rare TT (6-4) photoproducts are excised by cleavage seven and, less frequently, six phosphodiester bonds to the 5' side of the DNA lesion in addition to the primary cutting site at the eight 5' phosphodiester bond. The 3' cleavage sites are maintained at the fourth and fifth phosphodiester bonds for the these UV induced lesions. These data indicate that the cleavage pattern of the ABC excinuclease may be dependent upon both the type of DNA lesion as well as it surrounding nucleotide sequence. In addition, the authors analysis shows that (6-4) photoproducts are much better substrates for ABC excinuclease than are pyrimidine dimers

Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

Science.gov (United States)

Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

2006-01-01

RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

[Satellite RNA (RNA3) of tomato black ring virus is found with one of the 2 major RNAs (RNA2) in a new capsid nucleoprotein].

Science.gov (United States)

Doz, B; Dunez, J; Bove, J M

1977-12-19

Tomato Black Ring Virus (TBRV) like other NEPOviruses posseses two nucleoproteins M and B and two major RNAs, RNA1 and RNA2 respectively distributed in B and M. A new nucleoprotein has just been discovered and comprises one molecule of RNA2 associated with one molecule of RNA3. RNA3 is a small RNA of molecular weight 500,000 d considered to be a satellite RNA. Its level appears to depend on the infection stage, local or systemic. RNA3 is able to modify the relative proportions of nucleoproteins M and B and their respective RNAs. The satellite RNA, might be part of the genome and represent a monocistronic mRNA for protein capsid synthesis. However it seems perhaps more tempting to correlate TBRV-RNA3 with satellite RNA5 of certain strains of Cucumber mosaic virus.

NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state.

Science.gov (United States)

Wang, Jing; Bains, Henrietta; Anastasia, Agustin; Bracken, Clay

2018-04-01

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone 1 H, 13 C, and 15 N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2 M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.

Working with RNA

DEFF Research Database (Denmark)

Nielsen, Henrik

2011-01-01

Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

Novel Biomarker Discovery for Diagnostic and Therapeutic Strategies in Prostate Cancer

Science.gov (United States)

2015-06-01

Task 2: Prepare fluorinated RNA aptamer library for screening indolent and aggressive LNCaP cells. The pool of RNA aptamers is amplified from the...transcribed Fluorination of the nucleic acid backbone stabilizes the RNA aptamers against RNAse degradation. Status: completed. Task 3: Perform first...to metastasis-prone prostate cancer cell surface targets, and exert cell- specific toxicity . We propose that these aptamers may help to discriminate between progressive and indolent prostate cancer in clinical applications.

Co-LncRNA: investigating the lncRNA combinatorial effects in GO annotations and KEGG pathways based on human RNA-Seq data.

Science.gov (United States)

Zhao, Zheng; Bai, Jing; Wu, Aiwei; Wang, Yuan; Zhang, Jinwen; Wang, Zishan; Li, Yongsheng; Xu, Juan; Li, Xia

2015-01-01